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dsDNA BR Assay

1. Calculating the Concentration of Your Sample The Qubit 2 0 Fluorometer gives values for the Qubit dsDNA HS assay in ng mL This value corresponds to the concentration after your sample was diluted into the assay tube To calculate the concentration of your sample use the following equation Concentration of your sample QF value x 2 x where QF value the value given by the Qubit 2 0 Fluorometer x the number of microliters of sample you added to the assay tube This equation generates a result with the same units as the value given by the Qubit 2 0 Fluorometer i e if the Qubit 2 0 Fluorometer gave a cncentration in ng mL the result of the equation will be in ng mL Qubit dsDNA HS Assay Kit 4 Dilution Calculator 2 1 2 2 2 3 2 4 2 5 2 6 The Dilution Calculator feature of the Qubit 2 0 Fluorometer calculates the concentration of your original sample based on the volume of sample you have added to the assay tube To have the Qubit 2 0 Fluorometer perform this calculation for you follow the instruction below Upon completion of the sample measurement press Calculate Stock Conc The Dilution Calculator Screen containing the volume roller wheel is displayed Using the volume roller wheel select the volume of your original sample that you have added to the assay tube When you stop scrolling the Qubit 2 0 Fluorometer calculates the original sample concentration based on the measure
2. Photobleaching of the Qubit Reagent The Qubit reagents exhibit high photostability in the Qubit 2 0 Fluorometer showing lt 0 3 drop in fluorescence after 9 readings and lt 2 5 drop in fluorescence after 40 readings It is important to remember however that if the assay tube remains in the Qubit 2 0 Fluorometer for multiple readings a temporary reduction in fluorescence is observed as the solution increases in temperature see Appendix Figure 2 Note that the temperature inside the Qubit 2 0 Fluorometer may be as much as 3 C above room temperature after 1 hour For this reason if you want to perform multiple readings of a single tube remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading Calibrating the Qubit 2 0 Fluorometer For each assay you have the choice to run a new calibration or to use the values from the previous calibration As you first use the instrument you should perform a new calibration each time As you become familiar with the assays the instrument your pipetting accuracy and significant temperature fluctuations within your laboratory you should determine the level of comfort you have using the calibration data stored in from the last time the instrument was calibrated Remember also that the fluorescence signal in the tubes containing standards and the samples is stable for no longer than 30 minutes See Figure 1 in the Appendix for a
3. 5 ug mL to 10 ug mLt dsDNA useful for quantitation of genomic and miniprep DNA samples e accurate in the presence of RNA salts solvents proteins and free Qubit dsDNA BR Assay Kit Q32853 500 nucleotides core range high confidence 1 ng mL to 500 ng mLt extended ranges moderate confidence 0 5 ng mL to 1 ng mL and 500 ng mL to 600 ng mLt dsDNA e useful for quantitation of PCR products viral DNA and samples for subcloning Qubit dsDNA HS Assay Kit 032854 500 e accurate in the presence of RNA salts solvents proteins and free nucleotides Qubit dsDNA HS Assay Kit Q32851 100 core range high confidence 5 ng mL to 1 000 ng mLt extended ranges moderate confidence 1 ng mL to 5 ng mL and 1 000 ng mL to 1 200 ng mLt useful for quantitation of oligos primers denatured DNA PCR products accurate in the presence of salts urea solvents proteins ATP and agarose Qubit ssDNA Assay Kit Q10212 100 ssDNA core range high confidence 25 ng mL to 500 ng mLt extended ranges moderate confidence 20 ng mL to 25 ng mL and 500 ng mL to 1 000 ng mLt useful for quantitation of samples for microarray RT PCR and Northern blot procedures e accurate in the presence of DNA salts solvents proteins and free Qubit RNA Assay Kit Q32855 500 nucleotides Qubit RNA Assay Kit Q32852 100 RNA core range high confidence 0 1 ug mL to 5 ug mLt extended ranges moderate c
4. lt 4 C When stored as directed kits are stable for 6 months NA Not applicable Introduction The Qubit RNA Assay Kits for use with the Qubit 2 0 Fluorometer make RNA quantitation easy and accurate The kit provides concentrated assay reagent dilution buffer and prediluted RNA standards Simply dilute the reagent using the buffer provided add your sample any volume between 1 uL and 20 uL is acceptable and read the concentration using the Qubit 2 0 Fluorometer The assay is highly selective for RNA over double stranded DNA dsDNA Appendix Figure 1 and is accurate for initial sample concentrations from 250 pg uL to 100 ng L The assay is performed at room temperature and the signal is stable for 3 hours Common contaminants such as salts free nucleotides solvents detergents or protein are well tolerated in the assay Appendix Table 2 In addition to the Qubit RNA Assay Kits described here we also offer other kits for assaying DNA and protein Appendix Table 3 MAN0002327 Revised 1 October 2010 MP 32852 Before You Begin Materials Required but Not Provided Storing the Qubit RNA Assay Kits Critical Assay Parameters To determine the purity of your sample use the Qubit RNA Assay Kit together with the Qubit dsDNA BR Assay Kit Thease measurements will give you a much better indication of sample purity than that produced by an Ax 9 A g9 measurement To measure protein contam
5. 10011 830 Revised 1 October 2010 MP 10212 Storing the Qubit ssDNA Assay Kit Critical Assay Parameters Handling and Disposal The Qubit ssDNA reagent and buffer are designed for room temperature storage The Qubit ssDNA reagent is supplied in DMSO which freezes at temperatures lower than room temperature Store the ssDNA standards at 4 C Assay Temperature The Qubit ssDNA assay for the Qubit 2 0 Fluorometer delivers optimal performance when all solutions are at room temperature 22 28 C The Qubit assays are designed to be performed at room temperature as temperature fluctuations can influence the accuracy of the assay To minimize temperature fluctuations store the Qubit ssDNA reagent and the Qubit ssDNA buffer at room temperature and insert all assay tubes into the Qubit 2 0 Fluorometer only for as much time as it takes for the instrument to measure the fluorescence because the Qubit 2 0 Fluorometer can raise the temperature of the assay solution significantly even over a period of a few minutes Do not hold the assay tubes in your hand before reading as this warms the solution and results in a low reading Incubation Time To allow the Qubit assay to reach optimal fluorescence incubate the tubes for the DNA assays for 2 minutes after mixing the sample or standard with the working solution After this incubation period the fluorescence signal is stable for 30 minutes at room temperature
6. Each standard tube will require 190 uL of Qubit working solution and each sample tube will require anywhere from 180 uL to 199 uL Prepare sufficient Qubit working solution to accommodate all standards and samples For example for 8 samples prepare enough working solution for the samples and 2 standards 200 uL per tube in 10 tubes yields 2 mL of working solution 10 uL of Qubit reagent plus 1 990 uL of Qubit buffer Load 190 uL of Qubit working solution into each of the tubes used for standards Add 10 uL of each Qubit standard to the appropriate tube and mix by vortexing 2 3 seconds being careful not to create bubbles Note Careful pipetting is critical to ensure that exactly 10 uL of each Qubit dsDNA BR standard is added to 190 uL of Qubit working solution It is also important to label the lid of each standard tube correctly as calibration of the Qubit 2 0 Fluorometer requires that the standards be introduced to the instrument in the right order Load Qubit working solution into individual assay tubes so that the final volume in each tube after adding sample is 200 uL Note Your sample can be anywhere between 1 uL and 20 uL therefore load each assay tube with a volume of Qubit working solution anywhere between 180 uL and 199 uL Add each of your samples to assay tubes containing the correct volume of Qubit working solution prepared in step 1 6 and mix by vortexing 2 3 seconds The final vo
7. Figure 2 The temperature inside the Qubit 2 0 Fluorometer may be as much as 3 C above room temperature after 1 hour For this reason if you want to perform multiple readings of a single tube you should remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading Calibrating the Qubit 2 0 Fluorometer For each assay you have the choice to run a new calibration or to use the values from the previous calibration As you first use the instrument perform a new calibration each time As you become familiar with the assays the instrument your pipetting accuracy and significant temperature fluctuations within your laboratory you should determine the level of comfort you have using the calibration data stored in from the last time the instrument was calibrated Remember also that the fluorescence signal in the tubes containing standards and the samples is stable for no longer than 3 hours See Figure 3 in the Appendix for an example of the calibration curve used to generate the quantitation results Qubit Protein Assay Kits 2 Handling and Disposal Experimental Protocol 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 No data are available addressing the mutagenicity or toxicity of the Qubit protein reagent Component A This reagent is an organic dye and is provided as a solution in 1 2 propanediol Treat the Qubit protein reagent with the same safety pr
8. see Appendix Figure 2 Note that the temperature inside the Qubit 2 0 Fluorometer may be as much as 3 C above room temperature after 1 hour For this reason if you want to perform multiple readings of a single tube remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading Calibrating the Qubit 2 0 Fluorometer For each assay you have the choice to run a new calibration or to use the values from the previous calibration As you first use the instrument perform a new calibration each time As you become familiar with the assays the instrument your pipetting accuracy and significant temperature fluctuations within your laboratory determine the level of comfort you have using the calibration data stored in from the last time the instrument was calibrated Remember also that the fluorescence signal in the tubes containing standards and the samples is stable for not longer than 3 hours See Figure 3 in the Appendix for an example of the calibration curve used to generate the quantitation results Qubit RNA BR Assay Kits 2 RNAse free Handling Handling and Disposal Experimental Protocol Performing the Qubit RNA BR Assay 1 1 1 2 1 3 1 4 1 5 The calibration standards included in the Qubit RNA Assay Kit are high quality rRNA standards The integrity and concentration of these standards is critical to the optimal performance of the Qubit
9. 10 On the Standards Screen press Yes to run a new calibration or press No to use the last calibration 1 11 Ifyou pressed No on the Standards Screen proceed to step 1 12 If you selected Yes to a run new calibration follow instructions below Running a New Calibration Insert the tube containing Standard 1 in the Qubit 2 0 Fluorometer close the lid and press Read The reading will take approximately 3 seconds Remove Standard 1 Insert the tube containing Standard 2 in the Qubit 2 0 Fluorometer close the lid and press Read Remove Standard 2 1 12 If you pressed No on the Standards Screen the Sample Screen will be automatically displayed Insert a sample tube into the Qubit 2 0 Fluorometer close the lid and press Read 1 13 Upon the completion of the measurement the result will be displayed on the screen Note The value given by the Qubit 2 0 Fluorometer at this stage corresponds to the concentration after your sample was diluted into the assay tube You can record this value and perform the calculation yourself to find out the concentration of your original sample see Calculating the Concentration of Your Sample below or the Qubit 2 0 Fluorometer performs this calculation for you see Dilution Calculator next page 1 14 To read the next sample remove the sample from the Qubit 2 0 Fluorometer insert the next sample and press Read Next Sample 1 15 Repeat sample readings until all samples have been read
10. Assay Kit 100 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 00 cece cece cece een e eee eeee 1 kit Q32854 Qubit dsDNA HS Assay Kit 500 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee e cence eee eee ee 1 kit Q10212 Qubit ssDNA Assay Kit 100 assays 1 200 ng for use with the Qubit 2 0 Fluorometer 2 ce cece cece cence tent eee eeee 1 kit Q10210 Qubit RNA BR Assay Kit 100 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 0 c cece eee cence eee eens 1 kit Q10211 Qubit RNA BR Assay Kit 500 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 00 cee eee eee cence nee ee eens 1 kit Q32852 Qubit RNA Assay Kit 100 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 22 0 2 0 cece eee eee eect e een eeeneeee 1 kit Q32855 Qubit RNA Assay Kit 500 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee c eee e eee cent ene e en nenn 1 kit Q33211 Qubit Protein Assay Kit 100 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cece cece eee cent nee ee eens 1 kit Q33212 Qubit Protein Assay Kit 500 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cee eee eee e cee eee ene eee eeee 1 kit Q32856 Qubit assay tubes setof 500 en nen nd eben bahn 1 set Q32866 QubIt 2 0 Flusrometer e nn een name ne ande se han each Q
11. Q10211 Qubit RNA BR Assay Kit 500 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 00 cee eee ec cece e ene ene eee 1 kit Q32852 Qubit RNA Assay Kit 100 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 22 0 2 0 cece eee eee e eee eect nee en eens 1 kit Q32855 Qubit RNA Assay Kit 500 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee c eee e eee cent ene e en nenn 1 kit Q33211 Qubit Protein Assay Kit 100 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cece cece eee cent nee ee eens 1 kit Q33212 Qubit Protein Assay Kit 500 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cee eee eee e cee eee ene eee eeee 1 kit Q32856 Qubit assay tubes setof 500 en nen nd eben bahn 1 set Q32866 QubIt 2 0 Flusrometer e nn een name ne ande se han each Q32867 OQubit 2 0 Fluorometer USB ana ee ee ee eens each Q32868 Qubit 2 0 Fluorometer International Power Cord replacement 2224 ssesseesneneenennennneenenneeenenneennennnennnnn each Contact Information Molecular Probes Inc 29851 Willow Creek Road Eugene OR 97402 Phone 541 465 8300 Fax 541 335 0504 Customer Service 6 00 am to 4 30 pm Pacific Time Phone 541 335 0338 Fax 541 335 0305 probesorder invitrogen com Toll Free Ordering for USA Order Phone 800 438 2209 Order Fax 800 438 0228 Technical Service 8 00
12. SESH E REED EE een each Q32867 Oubit 2 0 Fluorometer USB anna ee ee ee ee each Q32868 Qubit 2 0 Fluorometer International Power Cord replacement 2224 ssesseeseeeseneneeneeeenenneeensenseenennsennnen each Contact Information Molecular Probes Inc 29851 Willow Creek Road Eugene OR 97402 Phone 541 465 8300 Fax 541 335 0504 Customer Service 6 00 am to 4 30 pm Pacific Time Phone 541 335 0338 Fax 541 335 0305 probesorder invitrogen com Toll Free Ordering for USA Order Phone 800 438 2209 Order Fax 800 438 0228 Technical Service 8 00 am to 4 00 pm Pacific Time Phone 541 335 0353 Toll Free 800 438 2209 Fax 541 335 0238 probestech invitrogen com Invitrogen European Headquarters Invitrogen Ltd 3 Fountain Drive Inchinnan Business Park Paisley PA4 ORF UK Phone 44 0 141 814 6100 Fax 44 0 141 814 6260 Email euroinfo invitrogen com Technical Services eurotech invitrogen com Further information on Molecular Probes products including product bibliographies is available from your local distributor or directly from Molecular Probes Customers in Europe Africa and the Middle East should contact our office in Paisley United Kingdom All others should contact our Technical Service Department in Eugene Oregon Molecular Probes products are high quality reagents and materials intended for research purposes only These products must be used by or directly under the s
13. a new calibration each time As you become familiar with the assays the instrument your pipetting accuracy and significant temperature fluctuations within your laboratory you should determine the level of comfort you have using the calibration data stored in from the last time the instrument was calibrated Remember also that the fluorescence signal in the tubes containing standards and the samples is stable for no longer than 3 hours See Figure 3 in the Appendix for an example of the calibration curve used to generate the quantitation results We must caution that no data are available addressing the mutagenicity or toxicity of the Qubit RNA reagent Component A This reagent is known to bind nucleic acid and is provided as a solution in DMSO Treat the Qubit RNA reagent with the same safety precautions as all other potential mutagens and dispose of the dye in accordance with local regulations The calibration standards included in the Qubit RNA Assay Kit are high quality rRNA standards The integrity and concentration of these standards is critical to the optimal performance of the Qubit RNA assay As such we highly recommend treating the rRNA standards as you would any other precious RNA Use appropriate RNAse free handling techniques including RNAse free gloves pipette tips and tubes Keep the tube lids closed whenever possible do not touch the pipet to the inside wall of the tube when withdrawing a sample and return the rRNA
14. assay requires 2 standards Note Use only thin wall clear 0 5 mL optical grade real time PCR tubes Acceptable tubes include Qubit assay tubes 500 tubes Invitrogen Cat no Q32856 or Axygen PCR 05 C tubes VWR part number 10011 830 Label the tube lids Make the Qubit working solution by diluting the Qubit RNA BR reagent 1 200 in Qubit RNA BR buffer Use a clean plastic tube each time you make Qubit working solution Do not mix the working solution in a glass container Note The final volume in each tube must be 200 uL Each standard tube requires 190 uL of Qubit working solution and each sample tube requires anywhere from 180 uL to 199 uL Prepare sufficient Qubit working solution to accommodate all standards and samples For example for 8 samples prepare enough working solution for the samples and 2 standards 200 uL per tube in 10 tubes yields 2 mL of working solution 10 uL of Qubit reagent plus 1 990 uL of Qubit buffer Load 190 uL of Qubit working solution into each of the tubes used for standards Add 10 uL of each Qubit RNA BR standard to the appropriate tube and mix by vortexing 2 3 seconds being careful not to create bubbles Note Careful pipetting is critical to ensure that exactly 10 uL of each Qubit RNA BR standard is added to 190 uL of Qubit working solution It is also important to label the lid of each standard tube correctly as calibration of the Qubit 2 0 Fluorometer
15. for microarray RT PCR and Northern blot procedures accurate in the presence of DNA salts solvents proteins and free Qubit RNA Assay Kit Q32855 500 nucleotides Qubit RNA BR Assay Kit Q10210 100 Qubit RNA BR Assay Kit Q10211 500 RNA core range high confidence 0 1 ug mL to 5 ug mLt extended ranges moderate confidence 0 05 ug mL to 0 1 g mL and 5 6 ug mLt useful for quantitation of samples for microarray RT PCR and Northern blot procedures accurate in the presence of DNA salts solvents proteins and free nucleotides Qubit Protein Assay Kit Q33211 100 Qubit Protein Assay Kit Q33212 500 protein core range high confidence 1 25 g mL to 25 ug mLt extended ranges moderate confidence 1 ug mL to 1 25 g mL and 25 ug mL to 26 ug mLt little protein to protein difference in signal accurate in the presence of DTT mercaptoethanol amino acids and DNA signal is stable for 3 hours Based on an assay volume of 200 uL tConcentration ranges refer to the concentration of sample after dilution in the assay tube Qubit Protein Assay Kits 8 ProductList current prices may be obtained from our website or from our Customer Service Department Cat no Product Name Unit Size 033211 Qubit Protein Assay Kit 100 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer ete 1 kit Q33212 Qubit Protein Assay Kit 500 assays 0 25 5 ug
16. for use with the Qubit 2 0 Fluorometer 0 cece cece cece e een e ences 1 kit Related products Q10210 Qubit RNA BR Assay Kit 100 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 0 cece eee eee ene eee e eee 1 kit Q10211 Qubit RNA BR Assay Kit 500 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 0 cece eee eee n eee eens 1 kit Q32852 Qubit RNA Assay Kit 100 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee cece teen ene eee eeee 1 kit Q32855 Qubit RNA Assay Kit 500 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 0 cece cece eee e eee cent ene e nn eeee 1 kit Q10212 Qubit ssDNA Assay Kit 100 assays 1 200 ng for use with the Qubit 2 0 Fluorometer 0 cece eee eee eee eee n een eens 1 kit Q32850 Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 00sec cece eee eee eee eee 1 kit Q32853 Qubit dsDNA BR Assay Kit 500 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0 c cece eee e cence nee e eens 1 kit Q32851 Qubit dsDNA HS Assay Kit 100 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee cence nee e eens 1 kit Q32854 Qubit dsDNA HS Assay Kit 500 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 0 e cece e eee eee eee e eee 1 kit Q32856 Qubit assay tubes
17. instruction below 2 1 Upon completion of the sample measurement press Calculate Stock Conc The Dilution Calculator Screen containing the volume roller wheel is displayed 2 2 Using the volume roller wheel select the volume of your original sample that you have added to the assay tube When you stop scrolling the Qubit 2 0 Fluorometer calculates the original sample concentration based on the measured assay concentration 2 3 To change the units in which the original sample concentration is displayed press ng mL A pop up window showing the current unit selection as indicated by an adjacent red dash opens 2 4 Select the unit for your original sample concentration by touching the desired unit in the unit selection pop up window To close the unit selection pop up window touch anywhere on the screen outside the pop up The Qubit 2 0 Fluorometer automatically converts the units to your selection once the unit selection pop up window is closed Note The unit button next to your sample concentration reflects the change in the units e g if you change the unit to pg uL the button will display pg L 2 5 To save the data from your calculation to the Qubit 2 0 Fluorometer press Save on the Dilution Calculator screen The last calculated value of your measurement will be saved as a CSV file and tagged with a time and date stamp 2 6 To exit the Dilution Calculator screen press any navigator button on the bottom of the screen o
18. neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 Several Molecular Probes products and product applications are covered by U S and foreign patents and patents pending All names containing the designation are registered with the U S Patent and Trademark Office The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Copyright 2010 Life Technologies Corporation All rights reserved This information is subject to change without notice For research use only Not intended for any animal or human therapeutic or diagnostic use Qubit dsDNA HS Assay Kit 9 invitrogen MOLECULAR PROBES Qubit ssDNA Assay Kit For use with the Qubit 2 0 Fluorometer Table 1 Contents and storage information Material Amount Concentration Storage Stability Qubit ssDNA Reagent Room temperature 9 250 pL 200X concentrate in DMSO Desiccate Component A Protect from light Qubit ssDNA Buffer Component B 50 mL Not applicabl
19. oO oO to oO L 0 5 10 15 20 25 30 Concentration of Protein ug mL Figure 3 The curve fitting algorithm used to determine concentration in the Qubit protein assay The Qubit 2 0 Fluorometer generates concentration data based on the relationship between the two standards used in calibration This plot shows the line corresponding to the curve fitting algorithm a modified Hill plot used in the calculation of concentration data for the Qubit protein assay For reference the positions of the standards and a set of data points from an actual experiment are shown superimposed onto the line demonstrating that the curve fitting algorithm gives accurate values for quantitation Qubit Protein Assay Kits 6 Contaminants Tolerated by the Qubit Protein Assay Table 2 Effect of contaminants in the Qubit protein assay tested over the range 1 25 ug mL to 25 ug mL Contaminant en in concentrationin Concentrationin Result TORY 20 pL Sample 10 pL Sample Sodium chloride 20 mM 200 mM 400 mM OK t Magnesium chloride 2mM 20mM 40 mM OK Potassium chloride 200 mM 200 mM 400 mM OK Calcium chloride 2mM 20 mM 40 mM OK Ammonium sulfate 5mM 50 mM 100 mM OKt DTT 1 mM 10 mM 20 mM OK t B Mercaptoethanol 1 mM 10 mM 20 mM OK EDTA 1 mM 10 mM 20 mM OK Sodium azide 1 mM 10 mM 20 mM OK HEPES pH 7 4 5 mM 50 mM 100 mM OK oe phosphate 5mM 50 mM 100 mM OK rsp su
20. or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that
21. reagents and materials intended for research purposes only These products must be used by or directly under the supervision of a technically qualified individual experienced in handling potentially hazardous chemicals Please read the Material Safety Data Sheet provided for each product other regulatory considerations may apply Limited Use Label License No 223 Labeling and Detection Technology For research use only Not intended for any animal or human therapeutic or diagnostic use The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration an
22. uL per tube in 10 tubes yields 2 mL of working solution 10 uL of Qubit reagent plus 1 990 uL of Qubit buffer Load 190 uL of Qubit working solution into each of the tubes used for standards Add 10 uL of each Qubit standard to the appropriate tube and mix by vortexing 2 3 seconds being careful not to create bubbles Note Careful pipetting is critical to ensure that exactly 10 uL of each Qubit protein standard is added to 190 uL of Qubit working solution It is also important to label the lid of each standard tube correctly as calibration of the Qubit 2 0 Fluorometer requires that the standards be introduced to the instrument in the right order Load Qubit working solution into individual assay tubes so that the final volume in each tube after adding sample is 200 uL Note Your sample can be anywhere between 1 uL and 20 uL therefore load each assay tube with a volume of Qubit working solution anywhere between 180 uL and 199 uL Add each of your samples to assay tubes containing the correct volume of Qubit working solution prepared in step 1 6 and mix by vortexing 2 3 seconds The final volume in each tube should be 200 uL Allow all tubes to incubate at room temperature for 15 minutes Qubit Protein Assay Kits 3 1 9 On the Home Screen of the Qubit 2 0 Fluorometer press Protein The Standards Screen is automatically displayed Note If you have already performed a calibration for the select
23. volume of Qubit working solution prepared in step 1 6 and mix by vortexing 2 3 seconds The final volume in each tube should be 200 uL Allow all tubes to incubate at room temperature for 2 minutes On the Home Screen of the Qubit 2 0 Fluorometer press DNA and then select ssDNA as the assay type The Standards Screen is automatically displayed Note If you have already performed a calibration for the selected assay Qubit 2 0 Fluorometer will prompt you to choose between reading new standards and using the previous calibration See Calibrating the Qubit Fluorometer above for calibration guidelines Qubit ssDNA Assay Kit 3 1 10 On the Standards Screen press Yes to run a new calibration or press No to use the last calibration 1 11 Ifyou pressed No on the Standards Screen proceed to step 1 12 If you selected Yes to a run new calibration follow instructions below Running a New Calibration Insert the tube containing Standard 1 in the Qubit 2 0 Fluorometer close the lid and press Read The reading will take approximately 3 seconds Remove Standard 1 Insert the tube containing Standard 2 in the Qubit 2 0 Fluorometer close the lid and press Read Remove Standard 2 1 12 If you pressed No on the Standards Screen the Sample Screen will be automatically displayed Insert a sample tube into the Qubit 2 0 Fluorometer close the lid and press Read 1 13 Upon the completion of the measurement the resul
24. 0 2 0 DNA ng Fluorescence A pA 0 20 40 60 80 100 DNA or RNA ng Figure 1 DNA selectivity and sensitivity of the Qubit dsDNA HS assay 032851 032854 Triplicate 10 uL samples of A DNA O E coli rRNA A ora 1 1 mixture of DNA and RNA W were assayed in the Qubit dsDNA HS assay Fluorescence was measured at 485 530 nm and plotted versus the mass of nucleic acid for the DNA alone or RNA alone or versus the mass of the DNA component in the 1 1 mixture The variation CV of replicate DNA determinations was lt 2 The inset a separate experiment with octuplicate determinations shows the extreme sensitivity of the assay for DNA Background fluorescence has not been subtracted Effect of Temperature on the Qubit dsDNA HS Assay 160 140 120 4 100 80 60 40 Relative fluorescence normalized to room temperature 20 0 10 15 20 25 30 35 Assay temperature C Figure 2 Plot of fluorescence vs temperature for the Qubit dsDNA HS assay The Qubit assays were designed to be performed at room temperature as temperature fluctuations can influence the accuracy of the assay Qubit dsDNA HS Assay Kit 6 How the Qubit 2 0 Fluorometer Calculates Concentration Contaminants Tolerated by the Qubit dsDNA HS Assay Relative fluorescence 120 Standards 100 4 Raw Data Curve Fitting Algorithm 80 60 4
25. 0 4 20 4 O 100 200 300 400 500 600 Concentration of dsDNA ng mL Figure 3 The curve fitting algorithm used to determine concentration in the Qubit dsDNA HS assay The Qubit 2 0 Fluorometer generates concentration data based on the relationship between the two standards used in calibration This plot shows the line corresponding to the curve fitting algorithm a modified Hill plot used in the calculation of concentration data for the Qubit dsDNA HS assay For reference the positions of the standards and a set of data points from an actual experiment are shown superimposed onto the line demonstrating that the curve fitting algorithm gives accurate values for quantitation Table 2 Effect of contaminants in the Qubit dsDNA HS assay tested over the range of 1 ng mL to 500 ng mL Final RR Ara Contaminant Concentration in concen en ne concentrationiin Result the Assay pL Sample 10 pL Sample Sodium chloride 50mM 500 mM 1M OK Magnesium chloride 5mM 50mM 100 mM OK t Sodium acetate 30 mM 300 mM 600 mM OK Ammonium acetate 50 mM 500 mM 1M OK Ethanol 1 10 20 OK Phenol 0 1 1 2 OK t Chloroform 1 10 20 OK SDS 0 01 0 1 0 2 OK Triton X 100 0 01 0 1 0 2 OK t dNTPs 100 uM 1mM 2mM OK BSA 10 mg mL 100 mg mL 200 mg mL OKt IgG 0 5 mg mL 5 mg mL 10 mg mL OK RNA 1X 1X 1X OK ssDNA 1X 1X 1X OK Oligos 1X 1X 1X OK DNA standards were assayed in t
26. 0 Fluorometer 0 e eee e cece eee n eee eens 1 kit Q33211 Qubit Protein Assay Kit 100 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cee cece eee cence teen eens 1 kit Q33212 Qubit Protein Assay Kit 500 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cee eee eee e eee e ene eee eee 1 kit Q32856 Qubit assay tub s setof 500 u een be be beein ball 1 set Q32866 QubIt 2 0 Flusrometer e sororis een name ne ande nenne bE DEERE EEEE SEERE TERESA each Q32867 OQubit 2 0 Fluorometer USB ana ee ee ee eens each Q32868 Qubit 2 0 Fluorometer International Power Cord replacement 2224 ssesseesneneenennennneenenneeenenneennennnennnnn each Contact Information Molecular Probes Inc 29851 Willow Creek Road Eugene OR 97402 Phone 541 465 8300 Fax 541 335 0504 Customer Service 6 00 am to 4 30 pm Pacific Time Phone 541 335 0338 Fax 541 335 0305 probesorder invitrogen com Toll Free Ordering for USA Order Phone 800 438 2209 Order Fax 800 438 0228 Technical Service 8 00 am to 4 00 pm Pacific Time Phone 541 335 0353 Toll Free 800 438 2209 Fax 541 335 0238 probestech invitrogen com Invitrogen European Headquarters Invitrogen Ltd 3 Fountain Drive Inchinnan Business Park Paisley PA4 ORF UK Phone 44 0 141 814 6100 Fax 44 0 141 814 6260 Email euroinfo invitrogen com Technical Services eurotec
27. 0 mM 200 mM OK t Ammonium acetate 10mM 100 mM 200 mM OK Na phosphate 5 mM 50 mM 100 mM OK Ethanol 1 10 20 OK Phenol 0 1 1 2 OK t Chloroform 0 2 2 4 OK SDS 0 01 0 1 0 2 NR Triton X 100 0 001 0 01 0 02 OK dNTPs 100 uM 1mM 2mM OK BSA 20 g mL 200 pg mL 400 ug mL OK IgG 10 pg mL 100 ug mL 200 pg mL OK ssDNA 1X 1X 1X OK Oligos 1X 1X 1X OK dsDNA 1X 1X 1X OK E coli rRNA standards were assayed in the presence or absence of contaminants at the indicated final concentrations Equivalent concentrations approximate in 20 uL or 10 uL sample volumes are also listed Results are given either as OK usually less than 10 perturbation or as NR not recommended t An acceptable result but with some distortion of the standard curve for best results add the same amount of contaminant to the standard samples Immiscible 8 A mixture of dATP dCTP dGTP and dTTP Qubit RNA Assay Kits 7 Qubit Assay Kits Compatible with the Qubit 2 0 Fluorometer A number of fluorescence based quantitation kits are available for use with the Qubit 2 0 Fluorometer Table 3 will help you choose a kit based on the target molecule being measured and the number of assays you require Table 3 Qubit Assay Kits for use with the Qubit 2 0 Fluorometer Product Cat no Number f Target Notes Assays Qubit dsDNA BR Assay Kit Q32850 100 core range high confidence 0 01 ug mL to 5 ug mLt extended range moderate confidence
28. 32867 OQubit 2 0 Fluorometer USB ana ee ee ee eens each Q32868 Qubit 2 0 Fluorometer International Power Cord replacement 2224 ssesseesneneenennennneenenneeenenneennennnennnnn each Contact Information Molecular Probes Inc 29851 Willow Creek Road Eugene OR 97402 Phone 541 465 8300 Fax 541 335 0504 Customer Service 6 00 am to 4 30 pm Pacific Time Phone 541 335 0338 Fax 541 335 0305 probesorder invitrogen com Toll Free Ordering for USA Order Phone 800 438 2209 Order Fax 800 438 0228 Technical Service 8 00 am to 4 00 pm Pacific Time Phone 541 335 0353 Toll Free 800 438 2209 Fax 541 335 0238 probestech invitrogen com Invitrogen European Headquarters Invitrogen Ltd 3 Fountain Drive Inchinnan Business Park Paisley PA4 ORF UK Phone 44 0 141 814 6100 Fax 44 0 141 814 6260 Email euroinfo invitrogen com Technical Services eurotech invitrogen com Further information on Molecular Probes products including product bibliographies is available from your local distributor or directly from Molecular Probes Customers in Europe Africa and the Middle East should contact our office in Paisley United Kingdom All others should contact our Technical Service Department in Eugene Oregon Molecular Probes products are high quality reagents and materials intended for research purposes only These products must be used by or directly under the supervision of a technica
29. NA O or a 1 1 mixture of RNA and DNA W were assayed in the Qubit RNA BR assay Fluorescence was measured at 630 660 nm and plotted versus the mass of nucleic acid for the RNA alone or DNA alone or versus the mass of the RNA component in the 1 1 mixture The variation CV of replicate RNA determinations was lt 10 The inset is an enlargement of the graph to show the sensitivity of the assay for RNA Background fluorescence has not been subtracted P 160 a T 140 do 22 120 35 27 100 vE S S 80 4 mo oz 60 29 EN 40 ro E 20 De O 0 10 15 20 25 30 Assay temperature C 35 Figure 2 Plot of fluorescence vs temperature for the Qubit RNA assay The Qubit assays were designed to be performed at room temperature as temperature fluctuations can influence the accuracy of the assay Qubit RNA BR Assay Kits 6 How the Qubit 2 0 Fluorometer Calculates Concentration 120 standards 100 4 O Raw Data Curve Fitting O 1 Relative fluorescence gt a oO 20 4 Concentration of RNA ug mL Figure 3 The curve fitting algorithm used to determine concentration in the Qubit RNA BR assay The Qubit 2 0 Fluorometer generates concentration data based on the relationship between the two standards used in calibration This plot shows the line corresponding to the curve fitting algorithm a modified Hill plot used in the calculation of co
30. NA Assay Kit Q10212 100 SONA useful for quantitation of oligos primers denatured DNA PCR products e accurate in the presence of salts urea solvents proteins ATP and agarose core range high confidence 25 ng mL to 500 ng mLt Qubit RNA Assay Kit Q32852 100 extended ranges moderate confidence 20 ng mL to 25 ng mL and 500 ng mL to 1 000 ng mLt RNA useful for quantitation of samples for microarray RT PCR and Northern blot procedures e accurate in the presence of DNA salts solvents proteins and free Qubit RNA Assay Kit Q32855 500 nucleotides core range high confidence 0 1 ug mL to 5 ug mLt Qubit RNA BR Assay Kit Q10210 100 extended ranges moderate confidence 0 05 ug mL to 0 1 ug mL and 5 6 ug mLt RNA e useful for quantitation of samples for microarray RT PCR and Northern blot procedures Qubit RNA BR Assay Kit Q10211 500 e accurate in the presence of DNA salts solvents proteins and free nucleotides Qubit Protein core range high confidence 1 25 g mL to 25 ug mLt Assay Kit Q33211 100 extended ranges moderate confidence 1 ug mL to 1 25 ug mL and 25 ug mL to 26 ug mLt protein little protein to protein difference in signal e accurate in the presence of DTT mercaptoethanol amino acids and i DNA on 033212 500 signal is stable for 3 hours Assay Kit Based on an assay volume of 200 uL tConcentration ranges refer to the concentration of sample after di
31. Qubit RNA BR Assay Kit Q10210 100 Qubit RNA BR Assay Kit Q10211 500 RNA core range high confidence 0 1 ug mL to 5 ug mLt extended ranges moderate confidence 0 05 ug mL to 0 1 g mL and 5 6 ug mLt useful for quantitation of samples for microarray RT PCR and Northern blot procedures accurate in the presence of DNA salts solvents proteins and free nucleotides Qubit Protein Assay Kit 033211 100 Qubit Protein Assay Kit 033212 500 protein core range high confidence 1 25 ug mL to 25 ug mLt extended ranges moderate confidence 1 ug mL to 1 25 g mL and 25 ug mL to 26 ug mLt little protein to protein difference in signal accurate in the presence of DTT B mercaptoethanol amino acids and DNA signal is stable for 3 hours Based on an assay volume of 200 uL tConcentration ranges refer to the concentration of sample after dilution in the assay tube Qubit ssDNA Assay Kit 7 ProductList current prices may be obtained from our website or from our Customer Service Department Cat no Product Name Unit Size 010212 Qubit ssDNA Assay Kit 100 assays 1 200 ng for use with the Qubit 2 0 Fluorometer ce cece ee cee eee e tenn eee e eee 1 kit Related products Q32850 Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0 e cece cece eee eee eee 1 kit Q32853 Qubit dsDNA BR Assay K
32. Qubit 2 0 Fluorometer Assay Manual 1 dsDNA BR Assay 2 10 2 dsDNA HS Assay 11 19 3 ssDNA Assay 20 27 4 Protein Assay 28 36 5 RNA Assay 37 45 6 RNA BR Assay 46 53 invitrogen MOLECULAR PROBES Qubit dsDNA BR Assay Kits For use with the Qubit 2 0 Fluorometer Table 1 Contents and storage information Material Amount Concentration Storage Stability Qubit dsDNA BR reagent 200X concentrate in Room temperature Component A 250 uL or 1 25 mL Desiccate Protect from light DMSO Qubit dsDNA BR buffer Component B 50 mL or 250 mL NA Room temperature When stored as directed kits are stable for 6 months Qubit dsDNA BR standard 1 Component C 1mLor5mL 0 ng uL in TE buffer Qubit dsDNA BR standard 2 Component D 1mLor5mL 100 ng uL in TE buffer NA Not applicable Introduction The Qubit dsDNA BR Assay Kits for use with the Qubit 2 0 Fluorometer make DNA quantitation easy and accurate The kit provides concentrated assay reagent dilution buffer and pre diluted DNA standards Simply dilute the reagent using the buffer provided add your sample any volume between 1 uL and 20 uL is acceptable and read the concentration using the Qubit 2 0 Fluorometer The assay is highly selective for double stranded DNA dsDNA over RNA Appendix Figure 1 and is accurate for initial sample concentration from 100 pg uL 1 000 ng uL The assay is
33. RNA BR assay As such we highly recommend treating the rRNA standards as you would any other precious RNA Use appropriate RNAse free handling techniques including RNAse free gloves pipette tips and tubes Keep the tube lids closed whenever possible do not touch the pipet to the inside wall of the tube when withdrawing a sample and return the rRNA standard to the refrigerator as soon as possible after use No data are currently available addressing the mutagenicity or toxicity of the Qubit RNA BR reagent Component A This reagent is known to bind nucleic acid and is provided as a solution in DMSO Treat the Qubit RNA BR reagent with the same safety precautions as all other potential mutagens and dispose of the dye in accordance with local regulations The protocol below assumes you are preparing standards for calibrating the Qubit 2 0 Fluorometer If you plan to use the last calibration performed on the instrument you need fewer tubes step 1 1 and less working solution step 1 3 More detailed instructions on the use of the Qubit 2 0 Fluorometer corresponding to steps 1 9 1 15 and 2 1 2 6 can be found in the user manual accompanying the instrument For sample purity determinations it is possible to use the Qubit 2 0 Fluorometer to calculate the amount of dsDNA and RNA in the same sample Simply perform each assay for your sample Set up the required number of 0 5 mL tubes you need for standards and samples The Qubit RNA BR
34. alculator screen The last calculated value of your measurement will be saved in the CSV file and tagged with a time and date stamp To exit the Dilution Calculator screen press any navigator button on the bottom of the screen or Read Next Sample Note When you navigate away from the Dilution Calculator screen the Qubit 2 0 Fluorometer saves the last values for the sample volume and the units in the Dilution Calculator screen only Returning to the Dilution Calculator screen displays these last selected values Qubit ssDNA Assay Kit 5 Appendix How the Qubit 2 0 Fluorometer Calculates Concentrations Contaminants Tolerated by the Qubit ssDNA Assay 120 standards 100 4 O Raw Data Curve Fitting Relative fluorescence 0 T T T T T 0 200 400 600 800 1000 1200 Concentration of ssDNA ng mL Figure 1 The curve fitting algorithm used to determine concentration in the Qubit ssDNA assay The Qubit 2 0 Fluorometer generates concentration data based on the relationship between the two standards used in calibration This plot shows the line corresponding to the curve fitting algorithm a modified Hill plot used in the calculation of concentration data for the Qubit ssDNA assay For reference the positions of the standards and a set of data points from an actual experiment are shown superimposed onto the line demonstrating that the curve fitting algorithm gives accurate values f
35. am to 4 00 pm Pacific Time Phone 541 335 0353 Toll Free 800 438 2209 Fax 541 335 0238 probestech invitrogen com Invitrogen European Headquarters Invitrogen Ltd 3 Fountain Drive Inchinnan Business Park Paisley PA4 ORF UK Phone 44 0 141 814 6100 Fax 44 0 141 814 6260 Email euroinfo invitrogen com Technical Services eurotech invitrogen com Further information on Molecular Probes products including product bibliographies is available from your local distributor or directly from Molecular Probes Customers in Europe Africa and the Middle East should contact our office in Paisley United Kingdom All others should contact our Technical Service Department in Eugene Oregon Molecular Probes products are high quality reagents and materials intended for research purposes only These products must be used by or directly under the supervision of a technically qualified individual experienced in handling potentially hazardous chemicals Please read the Material Safety Data Sheet provided for each product other regulatory considerations may apply Limited Use Label License No 223 Labeling and Detection Technology For research use only Not intended for any animal or human therapeutic or diagnostic use The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic
36. ance when all solutions are at room temperature 22 28 C The Qubit assays are designed to be performed at room temperature and temperature fluctuations can influence the accuracy of the assay Appendix Figure 2 To minimize temperature fluctuations store the Qubit RNA reagent and the Qubit RNA buffer at room temperature and insert all assay tubes into the Qubit 2 0 Fluorometer only for as much time as it takes for the instrument to measure the fluorescence because the Qubit 2 0 Fluorometer can raise the temperature of the assay solution significantly even over a period of a few minutes Do not hold the assay tubes in your hand before reading as this will warm the solution and result in a low reading Incubation Time To allow the Qubit assay to reach optimal fluorescence incubate the tubes for the DNA and RNA assays for 2 minutes after mixing the sample or standard with the working solution After this incubation period the fluorescence signal is stable for 3 hours at room temperature Photobleaching of the Qubit Reagent The Qubit reagents exhibit high photostability in the Qubit 2 0 Fluorometer showing lt 0 3 drop in fluorescence after 9 readings and lt 2 5 drop in fluorescence after 40 readings It is important to remember however that if the assay tube remains in the Qubit 2 0 Fluorometer for multiple readings a temporary reduction in fluorescence will be observed as the solution increases in temperature
37. ay is performed at room temperature and the signal is stable for 3 hours Common contaminants such as salts free nucleotides solvents detergents or protein are well tolerated in the assay Appendix Table 2 In addition to the Qubit dsDNA HS Assay Kits described here we offer other kits for assaying RNA protein and dsDNA at a higher concentration range Appendix Table 3 Note All Qubit assay kits can also be used with the Qubit 1 0 Fluorometer Revised 1 October 2010 MP 32851 Before You Begin Materials Required but Not Provided Storing the Qubit dsDNA HS Assay Kits Critical Assay Parameters e Plastic container disposable for mixing the Qubit working solution step 1 3 e Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 The Qubit dsDNA HS reagent and buffer are designed for room temperature storage The Qubit dsDNA HS reagent is supplied in DMSO which freezes at temperatures lower than room temperature Store the DNA standards at 4 C Assay Temperature The Qubit dsDNA HS assay for the Qubit 2 0 Fluorometer delivers optimal performance when all solutions are at room temperature 22 28 C The Qubit assays were designed to be performed at room temperature as temperature fluctuations can influence the accuracy of the assay Appendix Figure 2 To minimize temperature fluctuations store the Qubit dsDNA HS reagent and the Qubit
38. bject to change without notice For research use only Not intended for any animal or human therapeutic or diagnostic use Qubit dsDNA BR Assay Kits 9 invitrogen MOLECULAR PROBES Qubit dsDNA HS Assay Kits For use with the Qubit 2 0 Fluorometer Table 1 Contents and storage information Material Amount Concentration Storage Stability Qubit dsDNA HS Reagent Room temperature g 250 uL or 1 25 mL 200X concentrate in DMSO Desiccate Component A Protect from light Qubit dsDNA HS Buffer Component B 50 mL or 250 mL NA Room temperature When stored as directed kits are Qubit dsDNA HS Standard 1 Component C stable for 6 months 1mLor5 mL 0 ng uL in TE buffer e lt 4 C Qubit dsDNA HS Standard 2 Component D 1mLor5 mL 10 ng pL in TE buffer NA Not applicable Introduction MAN0002326 The Qubit dsDNA HS Assay Kits for use with the Qubit 2 0 Fluorometer make DNA quantitation easy and accurate The kit provides concentrated assay reagent dilution buffer and pre diluted DNA standards Simply dilute the reagent using the buffer provided add your sample any volume between 1 uL and 20 uL is acceptable and read the concentration using the Qubit 2 0 Fluorometer The assay is highly selective for double stranded DNA dsDNA over RNA Appendix Figure 1 and is accurate for initial sample concentrations from 10 pg uL to 100 ng uL The ass
39. ct or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 Several Molecular Probes products and product applications are covered by U S and foreign patents and patents pending All names containing the designation are registered with the U S Patent and Trademark Office The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Copyright 2010 Life Technologies Corporation All rights reserve
40. d This information is subject to change without notice For research use only Not intended for any animal or human therapeutic or diagnostic use Qubit ssDNA Assay Kit 8 invitrogen MOLECULAR PROBES Qubit Protein Assay Kits For use with the Qubit 2 0 Fluorometer Table 1 Contents and storage information Material Amount Concentration Storage Room temperature Qubit proteinreagent 300 uL or 1 5 mL 200X concentrate in 1 2 propanediol Desiccate Component A Protect from light Qubit protein buffer 60 mL or 300 mL Not applicable Room temperature Component B Qubit protein standard 1 1mLor5mL 0 ng uL in TE buffer with 2 mM sodium azide Component C UDI protein standard p2 1mLor5 mL 200 ng uL in TE buffer with 2 mM sodium azide lt 4 C Component D Qubit protein standard 3 1mLor5mL 400 ng L in TE buffer with 2 mM sodium azide Component E When stored as directed kits are stable for 6 months Introduction The Qubit Protein Assay Kits for use with the Qubit 2 0 Fluorometer make protein quantitation easy and accurate The kits provide concentrated assay reagent dilution buffer and pre diluted BSA standards Simply dilute the reagent using the buffer provided add your sample any volume between 1 uL and 20 uL is acceptable and read the concentration using the Qubit 2 0 Fluorometer The assay is accurate for initial sample concentrat
41. d as a CSV file and tagged with a time and date stamp To exit the Dilution Calculator screen press any navigator button on the bottom of the screen or Read Next Sample Note When you navigate away from the Dilution Calculator screen the Qubit 2 0 Fluorometer saves the last values for the sample volume and the units in the Dilution Calculator screen only Returning to the Dilution Calculator screen displays these last selected values Qubit dsDNA BR Assay Kits 5 Appendix Selectivity of the Qubit dsDNA BR Assay DNA RNA 1 1 DNA Fluorescence RNA AN 0 200 400 600 800 1000 DNA or RNA ng Figure 1 DNA selectivity and sensitivity of the Qubit dsDNA BR assay 032850 032853 Triplicate 10 uL samples of A DNA O E coli rRNA A or a 1 1 mixture of DNA and RNA W were assayed in the Qubit dsDNA BR assay Fluorescence was measured at 485 530 nm and plotted versus the mass of nucleic acid for the DNA alone or RNA alone or versus the mass of the DNA component in the 1 1 mixture The variation CV of replicate DNA determinations was lt 3 The inset a separate experiment with octuplicate determinations shows the sensitivity of the assay for DNA Background fluorescence has not been subtracted Effect of Temperature on the Qubit dsDNA BR Assay zT 160 5 3 140 oo Q 120 85 a 100 GE 993 80 2 ge 60 Zg SN 40 ro E 20 O 0 10 15 20 25 30 35 Assa
42. d assay concentration To change the units in which the original sample concentration is displayed press ng mL A pop up window showing the current unit selection as indicated by an adjacent red dash opens Select the unit for your original sample concentration by touching the desired unit in the unit selection pop up window To close the unit selection pop up window touch anywhere on the screen outside the pop up The Qubit 2 0 Fluorometer automatically converts the units to your selection once the unit selection pop up window is closed Note The unit button next to your sample concentration reflects the change in the units e g if you change the unit to pg L the button will display pg L To save the data from your calculation to the Qubit 2 0 Fluorometer press Save on the Dilution Calculator screen The last calculated value of your measurement will be saved as a CSV file and tagged with a time and date stamp To exit the Dilution Calculator Screen press any navigator button on the bottom of the screen or Read Next Sample Note When you navigate away from the Dilution Calculator screen the Qubit 2 0 Fluorometer saves the last values for the sample volume and the units in the Dilution Calculator screen only Returning to the Dilution Calculator screen displays these last selected values Qubit dsDNA HS Assay Kit 5 Appendix Selectivity of the Qubit dsDNA HS Assay DNA RNA 1 1 DNA 0 0 1
43. d may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 Several Molecular Probes products and product applications are covered by U S and foreign patents and patents pending All names containing the designation are registered with the U S Patent and Trademark Office The trademarks mentioned herein are the property of Life Tech
44. d the same amount of contaminant to the standard samples Immiscible A mixture of dATP dCTP dGTP and dTTP Qubit RNA BR Assay Kits 7 Qubit Assay Kits Compatible with the Qubit 2 0 Fluorometer Table 3 Qubit Assay Kits for use with the Qubit 2 0 Fluorometer A number of fluorescence based quantitation kits are available for use with the Qubit 2 0 Fluorometer Table 3 helps you choose a kit based on the target molecule being measured and the number of assays you require Product Cat no ns f Target Notes Qubit dsDNA BR Assay Kit Q32850 100 e core range high confidence 0 01 g mL to 5 ug mLt extended range moderate confidence 5 ug mL to 10 ug mLt dsDNA useful for quantitation of genomic and miniprep DNA samples e accurate in the presence of RNA salts solvents proteins and free Qubit dsDNA BR Assay Kit Q32853 500 nucleotides core range high confidence 1 ng mL to 500 ng mLt Qubit dsDNA HS Assay Kit 032851 100 extended ranges moderate confidence 0 5 ng mL to 1 ng mL and 500 ng mL to 600 ng mLt dsDNA useful for quantitation of PCR products viral DNA and samples for subcloning Qubit dsDNA HS Assay Kit 032854 500 e accurate in the presence of RNA salts solvents proteins and free nucleotides core range high confidence 5 ng mL to 1 000 ng mLt extended ranges moderate confidence 1 ng mL to 5 ng mL and er N 1 000 ng mL to 1 200 ng mLt SUE SSD
45. dsDNA HS buffer at room temperature and insert all assay tubes into the Qubit 2 0 Fluorometer only for as much time as it takes for the instrument to measure the fluorescence as the Qubit 2 0 Fluorometer can raise the temperature of the assay solution significantly even over a period of a few minutes Do not hold the assay tubes in your hand before reading as this will warm the solution and result in a low reading Incubation Time In order to allow the Qubit assay to reach optimal fluorescence incubate the tubes for the DNA and RNA assays for 2 minutes after mixing the sample or standard with the working solution After this incubation period the fluorescence signal is stable for 3 hours at room temperature Photobleaching of the Qubit Reagent The Qubit reagents exhibit high photostability in the Qubit 2 0 Fluorometer showing lt 0 3 drop in fluorescence after 9 readings and lt 2 5 drop in fluorescence after 40 readings It is important to remember however that if the assay tube remains in the Qubit 2 0 Fluorometer for multiple readings a temporary reduction in fluorescence will be observed as the solution increases in temperature see Appendix Figure 2 The temperature inside the Qubit 2 0 Fluorometer may be as much as 3 C above room temperature after 1 hour For this reason if you want to perform multiple readings of a single tube you should remove the tube from the instrument and let it equilibrate to room tempe
46. e assay tube To calculate the concentration of your sample use the following equation Concentration of your sample QF value x 2 x where QF value the value given by the Qubit 2 0 Fluorometer x the number of microliters of sample you added to the assay tube This equation generates a result with the same units as the value given by the Qubit 2 0 Fluorometer i e if the Qubit 2 0 Fluorometer gave a concentration in g mL the result of the equation will be in ug mL The Dilution Calculator feature of the Qubit 2 0 Fluorometer calculates the concentration of your original sample based on the volume of sample you have added to the assay tube To have the Qubit 2 0 Fluorometer perform this calculation for you follow the instruction below Upon completion of the sample measurement press Calculate Stock Conc The Dilution Calculator Screen containing the volume roller wheel is displayed Using the volume roller wheel select the volume of your original sample that you have added to the assay tube When you stop scrolling the Qubit 2 0 Fluorometer calculates the original sample concentration based on the measured assay concentration To change the units in which the original sample concentration is displayed press ng mL A pop up window showing the current unit selection as indicated by an adjacent red dash opens Select the unit for your original sample concentration by touching the desired unit in the unit sel
47. e e Room temperature When stored as directed kits are Qubit ssDNA Standard 1 Tmt O ng ul in TE buffer stable for 6 months Component C o lt 4 C P Do not freeze Qubit ssDNA Standard 2 Component D 1mL 20 ng uL in TE buffer Introduction Before You Begin Materials Required but Not Provided MAN0001988 The Qubit ssDNA Assay Kit for use with the Qubit 2 0 Fluorometer makes DNA quantitation easy and accurate The kit includes concentrated assay reagent dilution buffer and pre diluted DNA standards Simply dilute the reagent using the buffer provided add your sample any volume between 1 uL and 20 uL is acceptable and read the concentration using the Qubit 2 0 Fluorometer The assay is accurate for initial sample concentrations from 50 pg L to 200 ng uL providing an assay range of 1 200 ng The assay is performed at room temperature and the signal is stable for 30 minutes Common contaminants such as salts free nucleotides solvents detergents or protein are well tolerated in the assay Appendix Table 2 In addition to the Qubit ssDNA Assay Kits described here we also offer other kits for assaying RNA protein and dsDNA Appendix Table 3 Note All Qubit assay kits can also be used with the Qubit 1 0 Fluorometer e Plastic container disposable for mixing the Qubit working solution step 1 3 e Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no
48. e range high confidence 1 25 g mL to 25 ug mLt extended ranges moderate confidence 1 ug mL to 1 25 g mL and 25 ug mL to 26 ug mLt little protein to protein difference in signal accurate in the presence of DTT mercaptoethanol amino acids and DNA signal is stable for 3 hours Based on an assay volume of 200 uL tConcentration ranges refer to the concentration of sample after dilution in the assay tube Qubit dsDNA HS Assay Kit 8 ProductList current prices may be obtained from our website or from our Customer Service Department Cat no Product Name Unit Size 032851 Qubit dsDNA HS Assay Kit 100 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer A 1 kit 032854 Qubit dsDNA HS Assay Kit 500 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee e cence nee ee eens 1 kit Related products Q32850 Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 00sec cece cece ene eee eee 1 kit Q32853 Qubit dsDNA BR Assay Kit 500 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0 cee cece cece ene eee eee 1 kit Q10212 Qubit ssDNA Assay Kit 100 assays 1 200 ng for use with the Qubit 2 0 Fluorometer ce cece eee e cence eee eee ee eens 1 kit Q10210 Qubit RNA BR Assay Kit 100 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 0 cece eee eee een eee eens 1 kit
49. e with the Qubit 2 0 Fluorometer 00 cece e eee e cence nee e eens 1 kit Q10212 Qubit ssDNA Assay Kit 100 assays 1 200 ng for use with the Qubit 2 0 Fluorometer 0 2 0 0 ce cece eee e cece een e eee eeee 1 kit Q32850 Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0 c cece eee e cence nee e eens 1 kit Q32853 Qubit dsDNA BR Assay Kit 500 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0 0 cee eee eee cence nee ee eens 1 kit Q32851 Qubit dsDNA HS Assay Kit 100 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 00 cee ce eee eee ene eee eee 1 kit Q32854 Qubit dsDNA HS Assay Kit 500 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee cence nee e eens 1 kit Q33211 Qubit Protein Assay Kit 100 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cee eee cence eee e tenn ences 1 kit Q33212 Qubit Protein Assay Kit 500 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cee cee eee eee cence nee ee eens 1 kit Q32856 Qubit assay tub s setor 500 u een ee se beein ball 1 set Q32866 QubIt 2 0 FlUOTOMetEl sss sss cece tees sororis een nenne ne seabed oa nenne ee en each Q32867 Oubit 2 0 Fluorometer USB anna ee ee ee ee each Q32868 Qubit 2 0 Fluorometer International Power Cord replacement 2224 ssesseeseeeseneneeneee
50. ecautions as other materials with similar properties and dispose of the dye in accordance with local regulations The protocol below assumes you are preparing standards for calibrating the Qubit 2 0 Fluorometer If you plan to use the last calibration performed on the instrument you need fewer tubes step 1 1 and less working solution step 1 3 More detailed instructions on the use of the Qubit 2 0 Fluorometer corresponding to steps 1 9 1 15 and 2 1 2 6 can be found in the user manual accompanying the instrument Set up the number of 0 5 mL tubes you need for standards and samples The Qubit protein assay requires 3 standards Note Use only thin wall clear 0 5 mL optical grade real time PCR tubes Acceptable tubes include Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 Label the tube lids Make the Qubit working solution by diluting the Qubit protein reagent 1 200 in Qubit protein buffer Use a clean plastic tube each time you make Qubit working solution Do not mix the working solution in a glass container Note The final volume in each tube must be 200 uL Each standard tube requires 190 uL of Qubit working solution and each sample tube requires anywhere from 180 uL to 199 uL Prepare sufficient Qubit working solution to accommodate all standards and samples For example for 7 samples prepare enough working solution for the samples and 3 standards 200
51. ection pop up window To close the unit selection pop up window touch anywhere on the screen outside the pop up The Qubit 2 0 Fluorometer automatically converts the units to your selection once the unit selection pop up window is closed Note The unit button next to your sample concentration reflects the change in the units e g if you change the unit to pg L the button will display pg L To save the data from your calculation to the Qubit 2 0 Fluorometer press Save on the Dilution Calculator screen The last calculated value of your measurement will be saved in the CSV file and tagged with a time and date stamp To exit the Dilution Calculator Screen press any navigator button on the bottom of the screen or Read Next Sample Note When you navigate away from the Dilution Calculator screen the Qubit 2 0 Fluorometer saves the last values for the sample volume and the units in the Dilution Calculator screen only Returning to the Dilution Calculator screen displays these last selected values Qubit RNA BR Assay Kits 5 Appendix Selectivity of the Qubit RNA BR Assay Effect of Temperature on the Qubit RNA Assay 0 Fluorescence RNA or DNA ng 1 1 50 100 RNA RNA DNA DNA T 0 200 400 600 800 1000 RNA or DNA ng 1200 Figure 1 RNA selectivity and sensitivity of the Qubit RNA BR assay 010210 Q10211 Triplicate 10 uL samples of E coli rRNA A A D
52. ed assay Qubit 2 0 Fluorometer will prompt you to choose between reading new standards and using the previous calibration See Calibrating the Qubit 2 0 Fluorometer above for calibration guidelines 1 10 On the Standards Screen press Yes to run a new calibration or press No to use the last calibration 1 11 Ifyou pressed No on the Standards Screen proceed to step 1 12 If you selected Yes to a run new calibration follow instructions below Running a New Calibration Insert the tube containing Standard 1 in the Qubit 2 0 Fluorometer close the lid and press Read The reading will take approximately 3 seconds Remove Standard 1 Insert the tube containing Standard 2 in the Qubit 2 0 Fluorometer close the lid and press Read Remove Standard 2 Insert the tube containing Standard 3 in the Qubit 2 0 Fluorometer close the lid and press Read Remove Standard 3 1 12 If you pressed No on the Standards Screen the Sample Screen will be automatically displayed Insert a sample tube into the Qubit 2 0 Fluorometer close the lid and press Read 1 13 Upon the completion of the measurement the result will be displayed on the screen Note The value given by the Qubit 2 0 Fluorometer at this stage corresponds to the concentration after your sample was diluted into the assay tube You can record this value and perform the calculation yourself to find out the concentration of your original sample see Calculating the Concent
53. enenneeensenseenennsennnen each Contact Information Molecular Probes Inc 29851 Willow Creek Road Eugene OR 97402 Phone 541 465 8300 Fax 541 335 0504 Customer Service 6 00 am to 4 30 pm Pacific Time Phone 541 335 0338 Fax 541 335 0305 probesorder invitrogen com Toll Free Ordering for USA Order Phone 800 438 2209 Order Fax 800 438 0228 Technical Service 8 00 am to 4 00 pm Pacific Time Phone 541 335 0353 Toll Free 800 438 2209 Fax 541 335 0238 probestech invitrogen com Invitrogen European Headquarters nvitrogen Ltd 3 Fountain Drive nchinnan Business Park Paisley PA4 ORF UK Phone 44 0 141 814 6100 Fax 44 0 141 814 6260 Email euroinfo invitrogen com Technical Services eurotech invitrogen com Further information on Molecular Probes products including product bibliographies is available from your local distributor or directly from Molecular Probes Customers in Europe Africa and the Middle East should contact our office in Paisley United Kingdom All others should contact our Technical Service Department in Eugene Oregon Molecular Probes products are high quality reagents and materials intended for research purposes only These products must be used by or directly under the supervision of a technically qualified individual experienced in handling potentially hazardous chemicals Please read the Material Safety Data Sheet provided for each product other regulato
54. es om Stns mode required Sucrose 50 mM 500 mM 1M OK Sucrose 100 mM 1M 2M NR Glycerol 1 10 20 OK t Imidazole 1 25mM 12 5 mM 25 mM OK SDS 0 01 0 1 0 2 OK t SDS 0 02 0 2 0 4 NR Tween 20 0 001 0 01 0 02 NR Triton X 100 0 001 0 01 0 02 NR Amino acids 100 pg mL 1 mg mL 2 mg mL OK dNTPs 100 uM 1mM 2 mM OKt DNA 5 ug mL 50 ug mL 100 ug mL OKt DNA 10 tt 10 tt 10 tt OK DNA 50 tt 50 tt 50 tt NR BSA standards were assayed in the presence or absence of contaminants at the indicated final concentrations Equivalent concentrations approxima 10 perturbation or as NR e in 20 uL or 10 uL sample volumes are also listed Results are given as OK usually less than not recommended t An acceptable result but with some distortion of the standard curve For best results add the same amount of contaminant to the standard samples A precipitate was observed A mixture of 19 amino acids A mixture of dATP dCTP dGTP and dTTP tt For each data point the DNA mass was a fixed percentage of the protein mass For accurate results add the same amount of PBS to standard samples Qubit Protein Assay Kits 7 Qubit Assay Kits Compatible with the Qubit 2 0 Fluorometer Table 3 Qubit Assay Kits for use with the Qubit 2 0 Fluorometer A number of fluorescence based quantitation kits are available for use with the Qubit 2 0 Fluorometer Table 3 will help you choose a k
55. g solution Do not mix the working solution in a glass container Note The final volume in each tube must be 200 uL Each standard tube requires 190 uL of Qubit working solution and each sample tube requires anywhere from 180 uL to 199 uL Prepare sufficient Qubit working solution to accommodate all standards and samples For example for 8 samples prepare enough working solution for the samples and 2 standards 200 uL per tube in 10 tubes yields 2 mL of working solution 10 uL of Qubit reagent plus 1 990 uL of Qubit buffer Load 190 uL of Qubit working solution into each of the tubes used for standards Add 10 uL of each Qubit standard to the appropriate tube and mix by vortexing 2 3 seconds being careful not to create bubbles Note Careful pipetting is critical to ensure that exactly 10 uL of each Qubit ssDNA standard is added to 190 uL of Qubit working solution It is also important to label the lid of each standard tube correctly as calibration of the Qubit 2 0 Fluorometer requires that the standards are introduced to the instrument in the right order Load Qubit working solution into individual assay tubes so that the final volume in each tube after adding sample is 200 uL Note Your sample can be anywhere between 1 uL and 20 uL therefore load each assay tube with a volume of Qubit working solution anywhere between 180 uL and 199 uL Add each of your samples to assay tubes containing the correct
56. gh the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Molecular Pr
57. h invitrogen com Further information on Molecular Probes products including product bibliographies is available from your local distributor or directly from Molecular Probes Customers in Europe Africa and the Middle East should contact our office in Paisley United Kingdom All others should contact our Technical Service Department in Eugene Oregon Molecular Probes products are high quality reagents and materials intended for research purposes only These products must be used by or directly under the supervision of a technically qualified individual experienced in handling potentially hazardous chemicals Please read the Material Safety Data Sheet provided for each product other regulatory considerations may apply Limited Use Label License No 223 Labeling and Detection Technology For research use only Not intended for any animal or human therapeutic or diagnostic use The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made throu
58. he presence or absence of contaminants at the indicated final concentrations Equivalent in 20 uL or 10 uL sample volumes are also listed In all cases results are given as OK usually less than 10 perturbation t An acceptable result but with some distortion of the standard curve for best results add the same amount of contaminant to the standard samples Immiscible A mixture of dATP dCTP dGTP and dTTP concentrations approximate Qubit dsDNA HS Assay Kit 7 Qubit Assay Kits Compatible with the Qubit 2 0 Fluorometer Table 3 Qubit Assay Kits for use with the Qubit 2 0 Fluorometer A number of fluorescence based quantitation kits are available for use with the Qubit 2 0 Fluorometer Table 3 will help you choose a kit based on the target molecule being measured and the number of assays you require Product Cat no numberof Target Notes Assays Qubit dsDNA BR Assay Kit 032850 100 core range high confidence 0 01 ug mL to 5 ug mLt extended range moderate confidence 5 ug mL to 10 ug mLt dsDNA useful for quantitation of genomic and miniprep DNA samples e accurate in the presence of RNA salts solvents proteins and free Qubit dsDNA BR Assay Kit Q32853 500 nucleotides e core range high confidence 1 ng mL to 500 ng mLt Qubit dsDNA HS Assay Kit 032851 100 extended ranges moderate confidence 0 5 ng mL to 1 ng mL and 500 ng mL to 600 ng mLt dsDNA
59. ill be observed as the solution increases in temperature see Appendix Figure 2 The temperature inside the Qubit 2 0 Fluorometer may be as much as 3 C above room temperature after 1 hour For this reason if you want to perform multiple readings of a single tube you should remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading Calibrating the Qubit 2 0 Fluorometer For each assay you have the choice to run a new calibration or to use the values from the previous calibration As you first use the instrument you should perform a new calibration each time As you become familiar with the assays the instrument your pipetting accuracy and significant temperature fluctuations within your laboratory you should determine the level of comfort you have using the calibration data stored in from the last time the instrument was calibrated Remember also that the fluorescence signal in the tubes containing standards and the samples is stable for no longer than 3 hours See Figure 3 in the Appendix for an example of the calibration curve used to generate the quantitation results Qubit dsDNA BR Assay Kits 2 Handling and Disposal Experimental Protocol Performing the Qubit dsDNA BR Assay 1 1 1 2 1 3 1 4 1 5 1 6 1 7 We must caution that no data are available addressing the mutagenicity or toxicity ofthe Qubit dsDNA BR reagent Component A Th
60. ination in nucleic acid samples simply run 1 20 uL of the sample in the Qubit protein assay Note All Qubit assay kits can also be used with the Qubit 1 0 Fluorometer e Plastic container disposable for mixing the Qubit working solution step 1 3 e Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 The Qubit RNA reagent and buffer are designed for room temperature storage The Qubit RNA reagent is supplied in DMSO which freezes at temperatures lower than room temperature Store the RNA standards at 4 C The Qubit RNA reagent is sensitive to light Store the vial in the dark when not in use Assay Temperature The Qubit RNA assay for the Qubit 2 0 Fluorometer delivers optimal performance when all solutions are at room temperature 22 28 C The Qubit assays were designed to be performed at room temperature as temperature fluctuations can influence the accuracy of the assay Appendix Figure 2 To minimize temperature fluctuations store the Qubit RNA reagent and the Qubit RNA buffer at room temperature and insert all assay tubes into the Qubit 2 0 Fluorometer only for as much time as it takes for the instrument to measure the fluorescence because the Qubit 2 0 Fluorometer can raise the temperature of the assay solution significantly even over a period of a few minutes Do not hold the assay tubes in your hand before reading as this will warm the s
61. ions from 12 5 ug mL to 5 mg mL and exhibits low protein to protein variation Appendix Figure 1 The assay is performed at room temperature and the signal is stable for 3 hours Common contaminants such as reducing reagents DTT B mercaptoethanol salts free nucleotides amino acids solvents or DNA but not detergents are well tolerated in the assay although some very slight modifications for the procedure are required for other contaminants Appendix Table 2 In addition to the Qubit Protein Assay Kits described here we also offer other kits for assaying dsDNA and RNA Appendix Table 3 MAN0002349 Revised 1 October 2010 MP 33211 Before You Begin Materials Required but Not Provided Storing the Qubit Protein Assay Kits Critical Assay Parameters e Plastic container disposable for mixing the Qubit working solution step 1 3 e Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 The Qubit protein reagent and buffer are designed for room temperature storage The Qubit protein reagent is supplied in DMSO which freezes at temperatures lower than room temperature Store the protein standards at 4 C Assay Temperature The Qubit protein assay for the Qubit 2 0 Fluorometer delivers optimal performance when all solutions are at room temperature 22 28 C The Qubit assays is designed to be performed at room temperature as temperature fluctuation
62. is reagent is known to bind nucleic acid and is provided as a solution in DMSO Treat the Qubit dsDNA BR reagent with the same safety precautions as all other potential mutagens and dispose of the dye in accordance with local regulations The protocol below assumes you will be preparing standards for calibrating the Qubit 2 0 Fluorometer If you plan to use the last calibration performed on the instrument you will need fewer tubes step 1 1 and less working solution step 1 3 More detailed instructions on the use of the Qubit 2 0 Fluorometer corresponding to steps 1 9 1 15 and 2 1 2 6 can be found in the user manual accompanying the instrument For sample purity determinations it is possible to use the Qubit 2 0 Fluorometer to calculate the amount of dsDNA and RNA in the same sample Simply perform each assay for your sample Set up the number of 0 5 mL tubes you will need for standards and samples The Qubit dsDNA BR assay requires 2 standards Note Use only thin wall clear 0 5 mL PCR tubes Acceptable tubes include Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 Label the tube lids Make the Qubit working solution by diluting the Qubit dsDNA BR reagent 1 200 in Qubit dsDNA BR buffer Use a clean plastic tube each time you make Qubit working solution Do not mix the working solution in a glass container Note The final volume in each assay tube must be 200 uL
63. it Q32853 500 dsDNA core range high confidence 0 01 g mL to 5 ug mLt extended range moderate confidence 5 ug mL to 10 ug mLt useful for quantitation of genomic and miniprep DNA samples accurate in the presence of RNA salts solvents proteins and free nucleotides Qubit dsDNA HS Assay Kit Q32851 100 Qubit dsDNA HS Assay Kit Q32854 500 dsDNA core range high confidence 1 ng mL to 500 ng mLt extended ranges moderate confidence 0 5 ng mL to 1 ng mL and 500 ng mL to 600 ng mLt useful for quantitation of PCR products viral DNA and samples for subcloning accurate in the presence of RNA salts solvents proteins and free nucleotides Qubit ssDNA Assay Kit Qubit RNA Assay Kit Q10212 Q32852 100 100 Qubit RNA Assay Kit Q32855 500 ssDNA RNA core range high confidence 5 ng mL to 1 000 ng mLt extended ranges moderate confidence 1 ng mL to 5 ng mL and 1 000 ng mL to 1 200 ng mLt useful for quantitation of oligos primers denatured DNA PCR products accurate in the presence of salts urea solvents proteins ATP and agarose core range high confidence 25 ng mL to 500 ng mLt extended ranges moderate confidence 20 ng mL to 25 ng mL and 500 ng mL to 1 000 ng mLt useful for quantitation of samples for microarray RT PCR and Northern blot procedures accurate in the presence of DNA salts solvents proteins and free nucleotides
64. it RNA Assay Kit Q32852 100 RNA core range high confidence 0 1 ug mL to 5 ug mLt extended ranges moderate confidence 0 05 ug mL to 0 1 ug mL and 5 6 ug mLt useful for quantitation of samples for microarray RT PCR and Northern blot procedures Qubit RNA BR Assay Kit Q10211 500 e accurate in the presence of DNA salts solvents proteins and free nucleotides Qubit RNA BR Assay Kit Q10210 100 RNA core range high confidence 1 25 g mL to 25 ug mLt Q33211 100 extended ranges moderate confidence 1 ug mL to 1 25 ug mL and 25 ug mL to 26 ug mLt little protein to protein difference in signal e accurate in the presence of DTT B mercaptoethanol amino acids and DNA signal is stable for 3 hours Qubit Protein Assay Kit protein Qubit Protein Assay Kit Q33212 500 Based on an assay volume of 200 uL tConcentration ranges refer to the concentration of sample after dilution in the assay tube Qubit dsDNA BR Assay Kits 8 ProductList current prices may be obtained from our website or from our Customer Service Department Cat no Product Name Unit Size 032850 Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer tet 1 kit Q32853 Qubit dsDNA BR Assay Kit 500 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0 0 c cece eee cence nee ee eens 1 kit Related products Q32851 Qubit dsDNA HS
65. it 500 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0 e cece eee e eee een e ene eee 1 kit Q32851 Qubit dsDNA HS Assay Kit 100 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 00 cee cece eee cent eee eee 1 kit Q32854 Qubit dsDNA HS Assay Kit 500 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 0 cece cece cence nee e eens 1 kit Q10210 Qubit RNA BR Assay Kit 100 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 0 c cece eee cence teen eens 1 kit Q10211 Qubit RNA BR Assay Kit 500 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 00 cece e eee cence eee eens 1 kit Q32852 Qubit RNA Assay Kit 100 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee cence eee eee n eee nenn 1 kit Q32855 Qubit RNA Assay Kit 500 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 0 cece cece eee eee cent ene e en nenn 1 kit Q33211 Qubit Protein Assay Kit 100 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cece cece eee cent nee ee eens 1 kit Q33212 Qubit Protein Assay Kit 500 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cee eee eee e cence e een e eee eeee 1 kit Q32856 Qubit assay tubes setof 500 ent nen nn erben bahn 1 set Q32866 QubIt 2 0 FIUOTOMeCtEl sss sss deve tees sororis messen nenne ne ande oe EEEIEE
66. it based on the target molecule being measured and the number of assays you require Product Cat no numberof Target Notes Assays Qubit dsDNA BR Assay Kit 032850 100 core range high confidence 0 01 ug mL to 5 ug mLt extended range moderate confidence 5 ug mL to 10 ug mLt dsDNA useful for quantitation of genomic and miniprep DNA samples e accurate in the presence of RNA salts solvents proteins and free Qubit dsDNA BR Assay Kit Q32853 500 nucleotides e core range high confidence 1 ng mL to 500 ng mLt Qubit dsDNA HS Assay Kit 032851 100 extended ranges moderate confidence 0 5 ng mL to 1 ng mL and 500 ng mL to 600 ng mLt dsDNA useful for quantitation of PCR products viral DNA and samples for subcloning Qubit dsDNA HS Assay Kit 032854 500 e accurate in the presence of RNA salts solvents proteins and free nucleotides core range high confidence 5 ng mL to 1 000 ng mLt extended ranges moderate confidence 1 ng mL to 5 ng mL and Ben i 1 000 ng mL to 1 200 ng mLt Qube ssDNA Assay Kit Q10212 109 SSDNA useful for quantitation of oligos primers denatured DNA PCR products e accurate in the presence of salts urea solvents proteins ATP and agarose core range high confidence 25 ng mL to 500 ng mLt Qubit RNA Assay Kit Q32852 100 extended ranges moderate confidence 20 ng mL to 25 ng mL and 500 ng mL to 1 000 ng mLt RNA useful for quantitation of samples
67. ll tubes to incubate at room temperature for 2 minutes On the Home Screen of the Qubit 2 0 Fluorometer press RNA and then select RNA again as the assay type The Standards Screen is automatically displayed Note If you have already performed a calibration for the selected assay Qubit 2 0 Fluorometer will prompt you to choose between reading new standards and using the previous calibration See Calibrating the Qubit 2 0 Fluorometer above for calibration guidelines On the Standards Screen press Yes to run a new calibration or press No to use the last calibration If you pressed No on the Standards Screen proceed to step 1 12 If you selected Yes to a run new calibration follow instructions below Running a New Calibration Insert the tube containing Standard 1 in the Qubit 2 0 Fluorometer close the lid and press Read The reading will take approximately 3 seconds Remove Standard 1 Insert the tube containing Standard 2 in the Qubit 2 0 Fluorometer close the lid and press Read Remove Standard 2 If you pressed No on the Standards Screen the Sample Screen will be automatically displayed Insert a sample tube into the Qubit 2 0 Fluorometer close the lid and press Read Upon the completion of the measurement the result will be displayed on the screen Note The value given by the Qubit 2 0 Fluorometer at this stage corresponds to the concentration after your sample was diluted into the assay tube You can rec
68. lly qualified individual experienced in handling potentially hazardous chemicals Please read the Material Safety Data Sheet provided for each product other regulatory considerations may apply Limited Use Label License No 223 Labeling and Detection Technology For research use only Not intended for any animal or human therapeutic or diagnostic use The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to
69. lume in each tube should be 200 uL Qubit dsDNA BR Assay Kits 3 1 8 Allow all tubes to incubate at room temperature for 2 minutes 1 9 On the Home Screen of the Qubit 2 0 Fluorometer press DNA and then select dsDNA Broad Range as the assay type The Standards Screen is automatically displayed Note If you have already performed a calibration for the selected assay Qubit 2 0 Fluorometer will prompt you to choose between reading new standards and using the previous calibration See Calibrating the Qubit 2 0 Fluorometer above for calibration guidelines 1 10 On the Standards Screen press Yes to run a new calibration or press No to use the last calibration 1 11 Ifyou pressed No on the Standards Screen proceed to step 1 12 If you selected Yes to a run new calibration follow instructions below Running a New Calibration Insert the tube containing Standard 1 in the Qubit 2 0 Fluorometer close the lid and press Read The reading will take approximately 3 seconds Remove Standard 1 Insert the tube containing Standard 2 in the Qubit 2 0 Fluorometer close the lid and press Read Remove Standard 2 1 12 If you pressed No on the Standards Screen the Sample Screen will be automatically displayed Insert a sample tube into the Qubit 2 0 Fluorometer close the lid and press Read 1 13 Upon the completion of the measurement the result will be displayed on the screen Note The value given by the Qubit 2 0 Flu
70. lution in the assay tube Qubit RNA BR Assay Kits 8 ProductList current prices may be obtained from our website or from our Customer Service Department Cat no Product Name Unit Size 010210 Qubit RNA BR Assay Kit 100 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer A 1 kit 010211 Qubit RNA BR Assay Kit 500 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 0 c cece eee e cence nee e eens 1 kit Related products Q32852 Qubit RNA Assay Kit 100 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 2 1 02 0 cece eee ence eee e tenn e ee nenn 1 kit Q32855 Qubit RNA Assay Kit 500 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 00 cece cece eee e ee ene ene e en eeee 1 kit Q10212 Qubit ssDNA Assay Kit 100 assays 1 200 ng for use with the Qubit 2 0 Fluorometer 0 2 0 0 ce cece eee e cence een e eee eeee 1 kit Q32850 Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0 cece eee eee e ene eee e eee 1 kit Q32853 Qubit dsDNA BR Assay Kit 500 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 00 cee eee eee cence nee ee eens 1 kit Q32851 Qubit dsDNA HS Assay Kit 100 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer cee ce eee eee ene eee e eee 1 kit Q32854 Qubit dsDNA HS Assay Kit 500 assays 0 2 100 ng for use with the Qubit 2
71. n example of the calibration curve used to generate the quantitation results No data are currently available addressing the mutagenicity or toxicity of the Qubit ssDNA reagent Component A This reagent is known to bind nucleic acid and is provided as a solution in DMSO Treat the Qubit ssDNA reagent with the same safety precautions as all other potential mutagens and dispose of the dye in accordance with local regulations Qubit ssDNA Assay Kit 2 Experimental Protocol 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 The protocol below assumes you are preparing standards for calibrating the Qubit 2 0 Fluorometer If you plan to use the last calibration performed on the instrument you need fewer tubes step 1 1 and less working solution step 1 3 You can find more detailed instructions on the use of the Qubit 2 0 Fluorometer corresponding to steps 1 9 1 15 and 2 1 2 6 below in the user manual accompanying the instrument Set up the required number of 0 5 mL tubes for standards and samples The Qubit ssDNA assay requires 2 standards Note Use only thin wall clear 0 5 mL PCR tubes Acceptable tubes include Qubit assay tubes 500 tubes Invitrogen Cat no Q32856 or Axygen PCR 05 C tubes VWR part number 10011 830 Label the tube lids Make the Qubit working solution by diluting the Qubit ssDNA reagent 1 200 in Qubit ssDNA buffer Use a clean plastic tube each time you make Qubit workin
72. ncentration of your original sample based on the volume of sample you have added to the assay tube To have the Qubit 2 0 Fluorometer perform this calculation for you follow the instruction below Upon completion of the sample measurement press Calculate Stock Conc The Dilution Calculator Screen containing the volume roller wheel is displayed Using the volume roller wheel select the volume of your original sample that you have added to the assay tube When you stop scrolling the Qubit 2 0 Fluorometer calculates the original sample concentration based on the measured assay concentration To change the units in which the original sample concentration is displayed press ng mL A pop up window showing the current unit selection as indicated by an adjacent red dash opens Select the unit for your original sample concentration by touching the desired unit in the unit selection pop up window To close the unit selection pop up window touch anywhere on the screen outside the pop up The Qubit 2 0 Fluorometer automatically converts the units to your selection once the unit selection pop up window is closed Note The unit button next to your sample concentration reflects the change in the units e g if you change the unit to pg L the button will display pg L To save the data from your calculation to the Qubit 2 0 Fluorometer press Save on the Dilution Calculator screen The last calculated value of your measurement will be save
73. ncentration data for the Qubit RNA BR assay For reference the positions of the standards and a set of data points from an actual experiment are shown superimposed onto the line demonstrating that the curve fitting algorithm gives accurate values for quantitation Contaminants Tolerated by the Qubit RNA BR Assay Table 2 Effect of contaminants in the Qubit RNA BR assay tested over the range 500 5 000 ng mL Contaminant Final Concentration Concentration in Concentration in Result in the Assay 20 pL Sample 10 pL Sample Sodium chloride 10 mM 100 mM 200 mM OK Magnesium chloride 2 mM 20 mM 40 mM OKt Sodium acetate 10 mM 100 mM 200 mM OKt Ammonium acetate 10mM 100 mM 200 mM OK en phosphate 5 mM 50 mM 100 mM OK Ethanol 0 1 1 2 OK Phenol 0 1 1 2 Okt Chloroform 0 2 2 4 OK SDS 0 01 0 1 0 2 NR Triton X 100 0 001 0 01 0 02 OK dNTPs 100 uM 1mM 2 mM OK BSA 20 ug mL 200 ug mL 400 ug mL OK IgG 10 ug mL 100 ug mL 200 ug mL OK ssDNA 1X 1X 1X OK Oligos 1X 1X 1X OK dsDNA 1X 1X 1X OK E colirRNA standards were assayed in the presence or absence of contaminants at the indicated final concentrations Equivalent concentrations approximate in 20 uL or 10 uL sample volumes are also listed Results are given either as OK usually less than 10 perturbation or as NR not recommended tAn acceptable result but with some distortion of the standard curve for best results ad
74. nologies Corporation or their respective owners Copyright 2010 Life Technologies Corporation All rights reserved This information is subject to change without notice For research use only Not intended for any animal or human therapeutic or diagnostic use Qubit Protein Assay Kits 9 invitrogen Qubit RNA Assay Kits For use with the Qubit 2 0 Fluorometer Table 1 Contents and storage information MOLECULAR PROBES Material Amount Concentration Storage Stability Qubit RNA Assay Kit 100 assays Q32852 e Room temperature Qubit RNA Reagent Component A 250 uL 200 concentraten e Desiccate DMSO Protect from light N When stored Qubit RNA Buffer Component B 50 mL NA Room temperature as directed 1 tube kits are stable Qubit RNA Standard 1 Component C containing 1 mL 0 ng uL in TE buffer Qubit RNA Standard 2 Component D 4 tubes each containing 250 uL 10 ng pL in TE buffer for 6 months Qubit RNA Assay Kit 500 assays 032855 200X concentrate in Room temperature Qubit RNA Reagent Component A 1 25 mL Desiccate DMSO Protect from light Qubit RNA Buffer Component B 250 mL NA Room temperature 1 tube Qubit RNA Standard 1 Component C containing 5 mL 0 ng uL in TE buffer Qubit RNA Standard 2 Component D 10 tubes each containing 500 uL 10 ng uL in TE buffer
75. obes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 Several Molecular Probes products and product applications are covered by U S and foreign patents and patents pending All names containing the designation are registered with the U S Patent and Trademark Office The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Copyright 2010 Life Technologies Corporation All rights reserved This information is subject to change without notice For research use only Not intended for any animal or human therapeutic or diagnostic use Qubit RNA BR Assay Kits 9
76. olution and result in a low reading Incubation Time To allow the Qubit assay to reach optimal fluorescence incubate the tubes for the DNA and RNA assays for 2 minutes after mixing the sample or standard with the working solution After this incubation period the fluorescence signal is stable for 3 hours at room temperature Photobleaching of the Qubit Reagent The Qubit reagents exhibit high photostability in the Qubit 2 0 Fluorometer showing lt 0 3 drop in fluorescence after 9 readings and lt 2 5 drop in fluorescence after 40 readings It is important to remember however that if the assay tube remains in the Qubit 2 0 Fluorometer for multiple readings a temporary reduction in fluorescence will be observed as the solution increases in temperature see Appendix Figure 2 The temperature inside the Qubit 2 0 Fluorometer may be as much as 3 C above room temperature after 1 hour For this reason if you want to perform multiple readings of a single tube you should remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading Qubit RNA Assay Kits 2 Handling and Disposal Experimental Protocol Performing the Qubit RNA Assay 1 1 1 2 1 3 Calibrating the Qubit 2 0 Fluorometer For each assay you have the choice to run a new calibration or to use the values from the previous calibration As you first use the instrument you should perform
77. oncentration in Result in the Assay 20 uL Sample 10 pL Sample Sodium chloride 10 mM 100 mM 200 mM OKt Magnesium chloride 2mM 20 mM 40 mM OKt Sodium acetate 10 mM 100 mM 200 mM OK Ammonium acetate 10 mM 100 mM 200 mM OKt ras phosphate 5 mM 50 mM 100 mM OKt Ethanol 1 10 20 OK Phenol 0 1 1 2 OK Chloroform 0 2 2 4 OK SDS 0 01 0 1 0 2 OK Triton X 100 0 001 0 01 0 02 OKt dNTPs 100 uM 1mM 2mM OK BSA 20 ug mL 200 ug mL 400 ug mL OKt IgG 10 ug mL 100 g mL 200 pg mL OK RNA 6X 6X 6X OK ssDNA 1X 1X 1X OK Oligos 3X 3X 3X OK DNA standards were assayed in the presence or absence of contaminants at the indicated final concentrations Equivalent concentrations approximate in 20 uL or 10 uL sample volumes are also listed In all cases results are given as OK usually less than 10 perturbation t An acceptable result but with some distortion of the standard curve for best results add the same amount of contaminant to the standard samples Immiscible A mixture of dATP dCTP dGTP and dTTP Qubit dsDNA BR Assay Kits 7 Qubit Assay Kits Compatible with the Qubit 2 0 Fluorometer A number of fluorescence based quantitation kits are available for use with the Qubit 2 0 Fluorometer Table 3 will help you choose a kit based on the target molecule being measured and the number of assays you require Table 3 Qubit Assay Kits for use with the Qubit 2 0 Fluorometer Product Cat no Numbero i Target Notes A
78. onfidence 0 05 ug mL to 0 1 ug mL and 5 6 ug mLt e useful for quantitation of samples for microarray RT PCR and Northern blot procedures Qubit RNA BR Assay Kit Q10211 500 e accurate in the presence of DNA salts solvents proteins and free nucleotides Qubit RNA BR Assay Kit Q10210 100 RNA core range high confidence 1 25 g mL to 25 ug mLt Q33211 100 extended ranges moderate confidence 1 ug mL to 1 25 ug mL and 25 ug mL to 26 ug mLt little protein to protein difference in signal accurate in the presence of DTT mercaptoethanol amino acids and DNA signal is stable for 3 hours Qubit Protein Assay Kit protein Qubit Protein Assay Kit Q33212 500 Based on an assay volume of 200 uL tConcentration ranges refer to the concentration of sample after dilution in the assay tube Qubit RNA Assay Kits 8 Product List current prices may be obtained from our website or from our Customer Service Department Cat no Product Name Unit Size Q32852 Qubit RNA Assay Kit 100 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 1 kit Q32855 Qubit RNA Assay Kit 500 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 1 kit Related products Q10210 Qubit RNA BR Assay Kit 100 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 00 cece e eee cence nee ee eens 1 kit Q10211 Qubit RNA BR Assay Kit 500 assays 20 1000 ng for us
79. or quantitation Table 2 Effect of contaminants in the Qubit ssDNA assay Compound Maximum Acceptable Signal Change Concentration Salts Ammonium acetate 50 mM 13 decrease Sodium acetate 30 mM 3 decrease Sodium chloride 100 mM 25 decrease Zinc chloride 1mM 43 decrease Magnesium chloride 5 mM 34 decrease Urea 2M 47 increase Organic Solvents Phenol 0 2 19 decrease Ethanol 10 19 increase Chloroform 2 2 increase Detergents Sodium dodecyl sulfate 0 01 73 increase Triton X 100 0 1 11 increase Proteins Bovine serum albumin 2 20 increase IgG 0 1 37 decrease Other Compounds Polyethylene glycol 1 29 increase Agarose 0 1 8 increase ATP 0 1 30 increase The compounds were incubated at the indicated concentrations with Qubit ssDNA assay in the presence of 660 ng mL of a 24 mer M13 sequencing primer Qubit ssDNA Assay Kit 6 Qubit Assay Kits Compatible with the Qubit 2 0 Fluorometer Table 3 Qubit Assay Kits for use with the Qubit 2 0 Fluorometer A number of fluorescence based quantitation kits are available for use with the Qubit 2 0 Fluorometer Table 3 will help you choose a kit based on the target molecule being measured and the number of assays you require Product Cat no Number of Assays Target Notes Qubit dsDNA BR Assay Kit Q32850 100 Qubit dsDNA BR Assay K
80. ord this value and perform the calculation yourself to find out the concentration of your original sample see Calculating the Concentration of Your Sample next page or the Qubit 2 0 Fluorometer performs this calculation for you see Dilution Calculator next page To read the next sample remove the sample from the Qubit 2 0 Fluorometer insert the next sample and press Read Next Sample Repeat sample readings until all samples have been read Qubit RNA Assay Kits 4 Calculating the Concentration of Your Sample The Qubit 2 0 Fluorometer gives values for the Qubit RNA assay in ng mL This value corresponds to the concentration after your sample was diluted into the assay tube To calculate the concentration of your sample use the following equation Concentration of your sample QF value x 2 x where QF value the value given by the Qubit 2 0 Fluorometer x the number of microliters of sample you added to the assay tube This equation generates a result with the same units as the value given by the Qubit 2 0 Fluorometer i e if the Qubit 2 0 Fluorometer gave a concentration in ng mL the result of the equation will be in ng mL Dilution Calculator The Dilution Calculator feature of the Qubit 2 0 Fluorometer calculates the concentration of your original sample based on the volume of sample you have added to the assay tube To have the Qubit 2 0 Fluorometer perform this calculation for you follow the
81. orometer at this stage corresponds to the concentration after your sample was diluted into the assay tube You can record this value and perform the calculation yourself to find out the concentration of your original sample see Calculating the Concentration of Your Sample below or the Qubit 2 0 Fluorometer performs this calculation for you see Dilution Calculator next page 1 14 To read the next sample remove the sample from the Qubit 2 0 Fluorometer insert the next sample and press Read Next Sample 1 15 Repeat sample readings until all samples have been read Calculating the Concentration of Your Sample The Qubit 2 0 Fluorometer gives values for the Qubit dsDNA BR assay in g mL This value corresponds to the concentration after your sample was diluted into the assay tube To calculate the concentration of your sample use the following equation Concentration of your sample QF value x 280 x where QF value the value given by the Qubit 2 0 Fluorometer x the number of microliters of sample you added to the assay tube This equation generates a result with the same units as the value given by the Qubit 2 0 Fluorometer i e if the Qubit 2 0 Fluorometer gave a concentration in pg mL the result of the equation will be in ug mL Qubit dsDNA BR Assay Kits 4 Dilution Calculator 2 1 2 2 2 3 2 4 2 5 2 6 The Dilution Calculator feature of the Qubit 2 0 Fluorometer calculates the co
82. performed at room temperature and the signal is stable for 3 hours Common contaminants such as salts free nucleotides solvents detergents or protein are well tolerated in the assay Appendix Table 2 In addition to the Qubit dsDNA BR Assay Kits described here we offer other kits for assaying RNA protein and dsDNA at a lower concentration range Appendix Table 3 To determine the purity of your sample use the Qubit dsDNA BR Assay Kit together with the Qubit RNA Assay Kit Thease measurements will give you a much better indication of sample purity than that produced by an A260 A2s0 measurement To measure protein contamination in nucleic acid samples simply run 1 20 uL of the sample in the Qubit protein assay Note All Qubit assay kits can also be used with the Qubit 1 0 Fluorometer MAN0002325 Revised 1 October 2010 MP 32850 Before You Begin Materials Required but Not Provided Storing the Qubit dsDNA BR Assay Kits Critical Assay Parameters e Plastic container disposable for mixing the Qubit working solution e Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 The Qubit dsDNA BR reagent and buffer are designed for room temperature storage The Qubit dsDNA BR reagent is supplied in DMSO which freezes at temperatures lower than room temperature Store the DNA standards at 4 C Assay Temperature The Qubit dsDNA BR assay for the Q
83. provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 Several Molecular Probes products and product applications are covered by U S and foreign patents and patents pending All names containing the designation are registered with the U S Patent and Trademark Office The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Copyright 2010 Life Technologies Corporation All rights reserved This information is su
84. r Read Next Sample Note When you navigate away from the Dilution Calculator screen the Qubit 2 0 Fluorometer saves the last values for the sample volume and the units in the Dilution Calculator screen only Returning to the Dilution Calculator screen displays these last selected values Qubit RNA Assay Kits 5 Appendix Selectivity of the Qubit RNA Assay Effect of Temperature on the Qubit RNA Assay RNA RNA or DNA ng RNA DNA 1 1 Fluorescence DNA O O U 0 20 40 60 80 100 RNA or DNA ng Figure 1 RNA selectivity and sensitivity of the Qubit RNA assay Q32852 Q32855 Triplicate 10 uL samples of E colirRNA A DNA O or a 1 1 mixture of RNA and DNA W were assayed in the Qubit RNA assay Fluorescence was measured at 630 680 nm and plotted versus the mass of nucleic acid for the RNA alone or DNA alone or versus the mass of the RNA component in the 1 1 mixture The variation CV of replicate RNA determinations was lt 10 The inset is an enlargement of the graph to show the sensitivity of the assay for RNA Background fluorescence has not been subtracted 160 140 120 100 80 60 40 Relative fluorescence normalized to room temperature 20 0 4 T T T T 10 15 20 25 30 35 Assay temperature C Figure 2 Plot of fluorescence vs temperature for the Qubit RNA assay The Qubit assays were designed to be pe
85. r its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 Several Molecular Probes products and product applications are covered by U S and foreign patents and patents pending All names containing the designation are registered with the U S Patent and Trademark Office The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Copyright 2010 Life Technologies Corporation All rights reserved This information is subject to change without notice For research use only Not intended for any animal or human therapeutic or diagnostic use Qubit RNA Assay Kits 9 invit
86. ration of Your Sample below or the Qubit 2 0 Fluorometer performs this calculation for you see Dilution Calculator next page 1 14 To read the next sample remove the sample from the Qubit 2 0 Fluorometer insert the next sample and press Read Next Sample 1 15 Repeat sample readings until all samples have been read Calculating the Concentration of Your Sample The Qubit 2 0 Fluorometer gives values for the Qubit protein assay in g mL This value corresponds to the concentration after your sample was diluted into the assay tube To calculate the concentration of your sample use the following equation Concentration of your sample QF value x 2 x where QF value the value given by the Qubit 2 0 Fluorometer x the number of microliters of sample you added to the assay tube This equation generates a result with the same units as the value given by the Qubit 2 0 Fluorometer i e if the Qubit 2 0 Fluorometer gave a concentration in g mL the result of the equation will be in ug mL Qubit Protein Assay Kits 4 Dilution Calculator Appendix Protein to Protein Variation of the Qubit Protein Assay 2 1 2 2 2 3 2 4 2 5 2 6 Upon completion ofthe sample measurement press Calculate Stock Conc The Dilution Calculator Screen containing the volume roller wheel is displayed Using the volume roller wheel select the volume of your original sample that you have added to the assay tube When
87. rature for 30 seconds before taking another reading Calibrating the Qubit 2 0 Fluorometer For each assay you have the choice to run a new calibration or to use the values from the previous calibration As you first use the instrument you should perform a new calibration each time As you become familiar with the assays the instrument your pipetting accuracy and significant temperature fluctuations within your laboratory you should determine the level of comfort you have using the calibration data stored in from the last time the instrument was calibrated Remember also that the fluorescence signal in the tubes containing standards and the samples is stable for no longer than 3 hours See Figure 3 in the Appendix for an example of the calibration curve used to generate the quantitation results Qubit dsDNA HS Assay Kit 2 Handling and Disposal Experimental Protocol 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 We must caution that no data are available addressing the mutagenicity or toxicity of the Qubit dsDNA HS reagent Component A This reagent is known to bind nucleic acid and is provided as a solution in DMSO Treat the Qubit dsDNA HS reagent with the same safety precautions as all other potential mutagens and dispose of the dye in accordance with local regulations The protocol below assumes you will be preparing standards for calibrating the Qubit 2 0 Fluorometer If you plan to use the last calibration pe
88. requires that the standards be introduced to the instrument in the right order Qubit RNA BR Assay Kits 3 1 6 1 7 1 8 1 9 1 10 1 11 1 12 1 13 1 14 1 15 Load Qubit working solution into individual assay tubes so that the final volume in each tube after adding sample is 200 uL Note Your sample can be anywhere between 1 uL and 20 uL therefore load each assay tube with a volume of Qubit working solution anywhere between 180 uL and 199 uL Add each of your samples to assay tubes containing the correct volume of Qubit working solution prepared in step 1 6 and mix by vortexing 2 3 seconds The final volume in each tube should be 200 uL Allow all tubes to incubate at room temperature for 2 minutes On the Home Screen of the Qubit 2 0 Fluorometer press RNA and then select RNA Broad Range as the assay type The Standards Screen is automatically displayed Note If you have already performed a calibration for the selected assay Qubit 2 0 Fluorometer will prompt you to choose between reading new standards and using the previous calibration See Calibrating the Qubit 2 0 Fluorometer above for calibration guidelines On the Standards Screen press Yes to run a new calibration or press No to use the last calibration If you pressed No on the Standards Screen proceed to step 1 12 If you selected Yes to a run new calibration follow instructions below Running a New Calibration Insert the t
89. rformed at room temperature as temperature fluctuations can influence the accuracy of the assay Qubit RNA Assay Kits 6 How the Qubit 2 0 Fluorometer Calculates Concentration 120 Standards Raw Data Curve Fitting Algorithm Q oO fi 80 5 60 40 Relative fluorescence 20 0 100 200 300 400 500 600 Concentration of RNA ng mL Figure 3 The curve fitting algorithm used to determine concentration in the Qubit RNA assay The Qubit 2 0 Fluorometer generates concentration data based on the relationship between the two standards used in calibration This plot shows the line corresponding to the curve fitting algorithm a modified Hill plot used in the calculation of concentration data for the Qubit RNA assay For reference the positions of the standards and a set of data points from an actual experiment are shown superimposed onto the line demonstrating that the curve fitting algorithm gives accurate values for quantitation Contaminants Tolerated by the Rubles RNA ASSEy Table 2 Effect of contaminants in the Qubit RNA assay tested over the range 25 ng mL to 500 ng mL Contaminant Final Concentration Concentration in Concentration in Result in the Assay 20 uL Sample 10 pL Sample Sodium chloride 10 mM 100 mM 200 mM OK Magnesium chloride 2 mM 20 mM 40 mM OK t Sodium acetate 10 mM 10
90. rformed on the instrument you will need fewer tubes step 1 1 and less working solution step 1 3 More detailed instructions on the use of the Qubit 2 0 Fluorometer corresponding to steps 1 9 1 15 and 2 1 2 6 can be found in the user manual accompanying the instrument Set up the number of 0 5 mL tubes you will need for standards and samples The Qubit dsDNA HS assay requires 2 standards Note Use only thin wall clear 0 5 mL PCR tubes Acceptable tubes include Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 Label the tube lids Make the Qubit working solution by diluting the Qubit dsDNA HS reagent 1 200 in Qubit dsDNA HS buffer Use a clean plastic tube each time you make Qubit working solution Do not mix the working solution in a glass container Note The final volume in each tube must be 200 uL Each standard tube will require 190 uL of Qubit working solution and each sample tube will require anywhere from 180 uL to 199 uL Prepare sufficient Qubit working solution to accommodate all standards and samples For example for 8 samples prepare enough working solution for the samples and 2 standards 200 uL per tube in 10 tubes yields 2 mL of working solution 10 uL of Qubit reagent plus 1 990 uL of Qubit buffer Load 190 uL of Qubit working solution into each of the tubes used for standards Add 10 uL of each Qubit standard to the appropriate
91. rogen MOLECULAR PROBES Qubit RNA BR Assay Kits For use with the Qubit 2 0 Fluorometer Table 1 Contents and storage information Amount Material Q10210 Q10211 Concentration Storage Stability 100 assays 500 assays ete F 25 C Qubit RNA BR Reagent 250 pL 1 25 mL 200X concentrate in lt Desiccate Component A DMSO Protect from light Qubit RNA BR Buffer When stored Component B 50 mL 250 mL Not applicable lt 25 C as directed kits are stable for Qubit RNA BR Standard 1 1 mL 5 mL 0 ng ul in TE buffer 6 months Component C 2 6 C am Do not freeze Qubit RNA BR Standard 2 p Component D 4x 250 uL 10 x 500 uL 100 ng uL in TE buffer Introduction MAN0001987 The Qubit RNA BR Broad Range Assay Kits for use with the Qubit 2 0 Fluorometer make RNA quantitation easy and accurate The kit provides concentrated assay reagent dilution buffer and prediluted RNA standards Simply dilute the reagent using the buffer provided add your sample any volume between 1 uL and 20 uL is acceptable and read the concentration using the Qubit 2 0 Fluorometer The assay is highly selective for RNA over double stranded DNA dsDNA Appendix Figure 1 and is accurate for initial sample concentrations from 1 ng uL to 1 ug L providing an assay range from 20 1 000 ng The assay is performed at room temperature and the signal is stable for 3 hours Common contaminan
92. ry considerations may apply Limited Use Label License No 223 Labeling and Detection Technology For research use only Not intended for any animal or human therapeutic or diagnostic use The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product o
93. s can influence the accuracy of the assay Appendix Figure 2 To minimize temperature fluctuations store the Qubit protein reagent and the Qubit protein buffer at room temperature and insert all assay tubes into the Qubit 2 0 Fluorometer only for as much time as it takes for the instrument to measure the fluorescence because the Qubit 2 0 Fluorometer can raise the temperature of the assay solution significantly even over a period of a few minutes Do not hold the assay tubes in your hand before reading as this will warm the solution and result in a low reading Incubation Time To allow the Qubit protein assay to reach optimal fluorescence incubate the tubes for 15 minutes after mixing the sample or standard with the working solution After this incubation period the fluorescence signal is stable for 3 hours at room temperature For greatest accuracy of the protein assay the incubation time of the samples should be within 10 minutes of the incubation time of the standards Photobleaching of the Qubit Reagent The Qubit reagents exhibit high photostability in the Qubit 2 0 Fluorometer showing lt 0 3 drop in fluorescence after 9 readings and lt 2 5 drop in fluorescence after 40 readings It is important to remember however that if the assay tube remains in the Qubit 2 0 Fluorometer for multiple readings a temporary reduction in fluorescence will be observed as the solution increases in temperature see Appendix
94. setof 500 u nen ea een 1 set Q32866 Qubit 2 0 FlUOTOMeCtEl sssr cece desis nn nennen anne sees SETETE EEEIEE EREE AEE ee en each Q32867 Oubit 20 Fluorometer USB nn ee Pe sa Sees onan ehe each Q32868 Qubit 2 0 Fluorometer International Power Cord replacement 2224 ssesseesneneenennennneenenneeenenneennennnennnnn each Contact Information Molecular Probes Inc 29851 Willow Creek Road Eugene OR 97402 Phone 541 465 8300 Fax 541 335 0504 Customer Service 6 00 am to 4 30 pm Pacific Time Phone 541 335 0338 Fax 541 335 0305 probesorder invitrogen com Toll Free Ordering for USA Order Phone 800 438 2209 Order Fax 800 438 0228 Technical Service 8 00 am to 4 00 pm Pacific Time Phone 541 335 0353 Toll Free 800 438 2209 Fax 541 335 0238 probestech invitrogen com Invitrogen European Headquarters Invitrogen Ltd 3 Fountain Drive Inchinnan Business Park Paisley PA4 ORF UK Phone 44 0 141 814 6100 Fax 44 0 141 814 6260 Email euroinfo invitrogen com Technical Services eurotech invitrogen com Further information on Molecular Probes products including product bibliographies is available from your local distributor or directly from Molecular Probes Customers in Europe Africa and the Middle East should contact our office in Paisley United Kingdom All others should contact our Technical Service Department in Eugene Oregon Molecular Probes products are high quality
95. ssays core range high confidence 0 01 ug mL to 5 ug mLt extended range moderate confidence 5 ug mL to 10 ug mLt useful for quantitation of genomic and miniprep DNA samples accurate in the presence of RNA salts solvents proteins and free Qubit dsDNA BR Assay Kit Q32853 500 nucleotides Qubit dsDNA BR Assay Kit Q32850 100 dsDNA core range high confidence 1 ng mL to 500 ng mLt extended ranges moderate confidence 0 5 ng mL to 1 ng mL and 500 ng mL to 600 ng mLt useful for quantitation of PCR products viral DNA and samples for subcloning Qubit dsDNA HS Assay Kit Q32854 500 accurate in the presence of RNA salts solvents proteins and free nucleotides Qubit dsDNA HS Assay Kit Q32851 100 dsDNA core range high confidence 5 ng mL to 1 000 ng mLt extended ranges moderate confidence 1 ng mL to 5 ng mL and 1 000 ng mL to 1 200 ng mLt useful for quantitation of oligos primers denatured DNA PCR products accurate in the presence of salts urea solvents proteins ATP and agarose Qubit ssDNA Assay Kit Q10212 100 ssDNA core range high confidence 25 ng mL to 500 ng mLt extended ranges moderate confidence 20 ng mL to 25 ng mL and 500 ng mL to 1 000 ng mLt useful for quantitation of samples for microarray RT PCR and Northern blot procedures e accurate in the presence of DNA salts solvents proteins and free Qubit RNA Assay Kit Q32855 500 nucleotides Qub
96. standard to the refrigerator as soon as possible after use The protocol below assumes you will be preparing standards for calibrating the Qubit 2 0 Fluorometer If you plan to use the last calibration performed on the instrument you will need fewer tubes step 1 1 and less working solution step 1 3 More detailed instructions on the use of the Qubit 2 0 Fluorometer corresponding to numbered steps 1 9 1 16 below can be found in the user manual accompanying that instrument For sample purity determinations it is possible to use the Qubit 2 0 Fluorometer to calculate the amount of dsDNA and RNA in the same sample Simply perform each assay for your sample Set up the number of 0 5 mL tubes you will need for standards and samples The Qubit RNA assay requires 2 standards Note Use only thin wall clear 0 5 mL optical grade real time PCR tubes Acceptable tubes include Qubit assay tubes 500 tubes Invitrogen Cat no Q32856 or Axygen PCR 05 C tubes VWR part number 10011 830 Label the tube lids Make the Qubit working solution by diluting the Qubit RNA reagent 1 200 in Qubit RNA buffer Use a clean plastic tube each time you make Qubit working solution Do not mix the working solution in a glass container Note The final volume in each tube must be 200 uL Each standard tube will require 190 uL of Qubit working solution and each sample tube will require anywhere from 180 uL to 199 uL Prepare sufficient Q
97. t will be displayed on the screen Note The value given by the Qubit 2 0 Fluorometer at this stage corresponds to the concentration after your sample was diluted into the assay tube You can record this value and perform the calculation yourself to find out the concentration of your original sample see Calculating the Concentration of Your Sample below or the Qubit 2 0 Fluorometer performs this calculation for you see Dilution Calculator next page 1 14 To read the next sample remove the sample from the Qubit 2 0 Fluorometer insert the next sample and press Read Next Sample 1 15 Repeat sample readings until all samples have been read Calculating the Concentration of Your Sample The Qubit 2 0 Fluorometer gives values for the Qubit ssDNA assay in ng mL This value corresponds to the concentration after your sample was diluted into the assay tube To calculate the concentration of your sample use the following equation Concentration of your sample QF value x 700 x where QF value the value given by the Qubit 2 0 Fluorometer x the number of microliters of sample you added to the assay tube This equation generates a result with the same units as the value given by the Qubit 2 0 Fluorometer i e if the Qubit 2 0 Fluorometer gave a concentration in ng mL the result of the equation will be in ng mL Qubit ssDNA Assay Kit 4 Dilution Calculator 2 1 2 2 2 3 2 4 2 5 2 6 The Dil
98. ts such as salts free nucleotides solvents detergents or protein are well tolerated in the assay Appendix Table 2 In addition to the Qubit RNA BR Assay Kits described here we also offer other kits for assaying DNA and protein Appendix Table 3 To determine the purity of your sample use the Qubit RNA BR Assay Kit together with the Qubit dsDNA BR Assay Kit These measurements give you a much better indication of sample purity than that produced by measuring the Ay 9 A gp ratio To measure protein contamination in nucleic acid samples simply run 1 20 uL of the sample in the Qubit protein assay Note All Qubit assay kits can also be used with the Qubit 1 0 Fluorometer Revised 1 October 2010 MP 10210 Before You Begin Materials Required but Not Provided Storing the Qubit RNA Assay Kits Critical Assay Parameters e Plastic container disposable for mixing the Qubit working solution step 1 3 e Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 The Qubit RNA reagent and buffer are designed for room temperature storage The Qubit RNA reagent is supplied in DMSO which freezes at temperatures lower than room temperature Store the RNA standards at 4 C The Qubit RNA reagent is sensitive to light Store the vial in the dark when not in use Assay Temperature The Qubit RNA assay for the Qubit 2 0 Fluorometer delivers optimal perform
99. tube and mix by vortexing 2 3 seconds being careful not to create bubbles Note Careful pipetting is critical to ensure that exactly 10 uL of each Qubit dsDNA HS standard is added to 190 uL of Qubit working solution It is also important to label the lid of each standard tube correctly as calibration of the Qubit 2 0 Fluorometer requires that the standards be introduced to the instrument in the right order Load Qubit working solution into individual assay tubes so that the final volume in each tube after adding sample is 200 uL Note Your sample can be anywhere between 1 uL and 20 uL therefore load each assay tube with a volume of Qubit working solution anywhere between 180 uL and 199 uL Add each of your samples to assay tubes containing the correct volume of Qubit working solution prepared in step 1 6 and mix by vortexing 2 3 seconds The final volume in each tube should be 200 uL Allow all tubes to incubate at room temperature for 2 minutes Qubit dsDNA HS Assay Kit 3 1 9 On the Home Screen of the Qubit 2 0 Fluorometer press DNA and then select dsDNA High Sensitivity as the assay type The Standards Screen is automatically displayed Note If you have already performed a calibration for the selected assay Qubit 2 0 Fluorometer will prompt you to choose between reading new standards and using the previous calibration See Calibrating the Qubit 2 0 Fluorometer above for calibration guidelines 1
100. ube containing Standard 1 in the Qubit 2 0 Fluorometer close the lid and press Read The reading will take approximately 3 seconds Remove Standard 1 Insert the tube containing Standard 2 in the Qubit 2 0 Fluorometer close the lid and press Read Remove Standard 2 If you pressed No on the Standards Screen the Sample Screen will be automatically displayed Insert a sample tube into the Qubit 2 0 Fluorometer close the lid and press Read Upon the completion of the measurement the result will be displayed on the screen Note The value given by the Qubit 2 0 Fluorometer at this stage corresponds to the concentration after your sample was diluted into the assay tube You can record this value and perform the calculation yourself to find out the concentration of your original sample see Calculating the Concentration of Your Sample next page or the Qubit 2 0 Fluorometer performs this calculation for you see Dilution Calculator next page To read the next sample remove the sample from the Qubit 2 0 Fluorometer insert the next sample and press Read Next Sample Repeat sample readings until all samples have been read Qubit RNA BR Assay Kits 4 Calculating the Concentration of Your Sample Dilution Calculator 2 1 2 2 2 3 2 4 2 5 2 6 The Qubit 2 0 Fluorometer gives values for the Qubit RNA BR assay in ug mL This value corresponds to the concentration after your sample was diluted into th
101. ubit working solution to accommodate all standards and samples For example for 8 samples prepare enough working solution for the samples and 2 standards 200 uL per tube in 10 tubes yields 2 mL of working solution 10 uL of Qubit reagent plus 1 990 uL of Qubit buffer Qubit RNA Assay Kits 3 1 4 1 5 1 6 1 7 1 8 1 9 1 10 1 11 1 12 1 13 1 14 1 15 Load 190 uL of Qubit working solution into each of the tubes used for standards Add 10 uL of each Qubit standard to the appropriate tube and mix by vortexing 2 3 seconds being careful not to create bubbles Note Careful pipetting is critical to ensure that exactly 10 uL of each Qubit RNA standard is added to 190 uL of Qubit working solution It is also important to label the lid of each standard tube correctly as calibration of the Qubit 2 0 Fluorometer requires that the standards be introduced to the instrument in the right order Load Qubit working solution into individual assay tubes so that the final volume in each tube after adding sample is 200 uL Note Your sample can be anywhere between 1 uL and 20 uL therefore load each assay tube with a volume of Qubit working solution anywhere between 180 uL and 199 uL Add each of your samples to assay tubes containing the correct volume of Qubit working solution prepared in step 1 6 and mix by vortexing 2 3 seconds The final volume in each tube should be 200 uL Allow a
102. ubit 2 0 Fluorometer delivers optimal performance when all solutions are at room temperature 22 28 C The Qubit assays were designed to be performed at room temperature as temperature fluctuations can influence the accuracy of the assay Appendix Figure 2 To minimize temperature fluctuations store the Qubit dsDNA BR reagent and the Qubit dsDNA BR buffer at room temperature and insert all assay tubes into the Qubit 2 0 Fluorometer only for as much time as it takes for the instrument to measure the fluorescence because the Qubit 2 0 Fluorometer can raise the temperature of the assay solution significantly even over a period of a few minutes Do not hold the assay tubes in your hand before reading as this will warm the solution and result in a low reading Incubation Time To allow the Qubit assay to reach maximum fluorescence incubate the tubes for the DNA and RNA assays for 2 minutes after mixing the sample or standard with the working solution After this incubation period the fluorescence signal is stable for 3 hours at room temperature Photobleaching of the Qubit Reagent The Qubit reagents exhibit high photostability in the Qubit 2 0 Fluorometer showing lt 0 3 drop in fluorescence after 9 readings and lt 2 5 drop in fluorescence after 40 readings It is important to remember however that if the assay tube remains in the Qubit 2 0 Fluorometer for multiple readings a temporary reduction in fluorescence w
103. umin Lysozyme Casein Myoglobin a Casein Pepsin IgG Histone LEE OEMS Fluorescence 0 0 1 0 2 0 3 0 4 0 5 0 Protein ug Figure 1 Low protein to protein variation in the Qubit protein assay Q33211 Q33212 Solutions of the following proteins were prepared diluted and assayed in the Qubit protein assay bovine serum albumin BSA chicken egg ovalbumin chicken egg lysozyme bovine milk B casein equine myoglobin bovine milk a casein porcine pepsin mouse immunoglobulin IgG and calf thymus histone Fluorescence was measured at 485 590 nm and plotted versus the mass of the protein sample At 3 yg the fluorescence variation was 12 4 or 8 7 excluding the basic histone protein Background fluorescence has not been subtracted Qubit Protein Assay Kits 5 Effect of Temperature on the Qubit Protein Assay T 160 4409 w 140 4 g o 22 120 35 2 100 4 GE 8 80 TO Q 60 g oN 40 co E 20 e amp 0 r r r r 10 15 20 25 30 35 Assay temperature C Figure 2 Plot of fluorescence vs temperature for the Qubit protein assay The Qubit assays were designed to be performed at room temperature as temperature fluctuations can influence the accuracy of the assay How the Qubit 2 0 Fluorometer Calculates Concentration 120 Standards 100 4 Raw Data Curve Fitting Algorithm L Relative fluorescence gt O
104. upervision of a technically qualified individual experienced in handling potentially hazardous chemicals Please read the Material Safety Data Sheet provided for each product other regulatory considerations may apply Limited Use Label License No 223 Labeling and Detection Technology For research use only Not intended for any animal or human therapeutic or diagnostic use The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the produ
105. useful for quantitation of PCR products viral DNA and samples for subcloning Qubit dsDNA HS Assay Kit 032854 500 e accurate in the presence of RNA salts solvents proteins and free nucleotides core range high confidence 5 ng mL to 1 000 ng mLt extended ranges moderate confidence 1 ng mL to 5 ng mL and Ben i 1 000 ng mL to 1 200 ng mLt Qube ssDNA Assay Kit Q10212 109 SSDNA useful for quantitation of oligos primers denatured DNA PCR products e accurate in the presence of salts urea solvents proteins ATP and agarose core range high confidence 25 ng mL to 500 ng mLt Qubit RNA Assay Kit Q32852 100 extended ranges moderate confidence 20 ng mL to 25 ng mL and 500 ng mL to 1 000 ng mLt RNA useful for quantitation of samples for microarray RT PCR and Northern blot procedures accurate in the presence of DNA salts solvents proteins and free Qubit RNA Assay Kit Q32855 500 nucleotides Qubit RNA BR Assay Kit Q10210 100 Qubit RNA BR Assay Kit Q10211 500 RNA core range high confidence 0 1 ug mL to 5 ug mLt extended ranges moderate confidence 0 05 ug mL to 0 1 g mL and 5 6 ug mLt useful for quantitation of samples for microarray RT PCR and Northern blot procedures accurate in the presence of DNA salts solvents proteins and free nucleotides Qubit Protein Assay Kit Q33211 100 Qubit Protein Assay Kit Q33212 500 protein cor
106. ution Calculator feature of the Qubit 2 0 Fluorometer calculates the concentration of your original sample based on the volume of sample you have added to the assay tube To have the Qubit 2 0 Fluorometer perform this calculation for you follow the instruction below Upon completion of the sample measurement press Calculate Stock Conc The Dilution Calculator Screen containing the volume roller wheel is displayed Using the volume roller wheel select the volume of your original sample that you have added to the assay tube When you stop scrolling the Qubit 2 0 Fluorometer calculates the original sample concentration based on the measured assay concentration To change the units in which the original sample concentration is displayed press ng mL A pop up window showing the current unit selection as indicated by an adjacent red dash opens Select the unit for your original sample concentration by touching the desired unit in the unit selection pop up window To close the unit selection pop up window touch anywhere on the screen outside the pop up The Qubit 2 0 Fluorometer automatically converts the units to your selection once the unit selection pop up window is closed Note The unit button next to your sample concentration reflects the change in the units e g if you change the unit to pg L the button will display pg L To save the data from your calculation to the Qubit 2 0 Fluorometer press Save on the Dilution C
107. y temperature C Figure 2 Plot of fluorescence vs temperature for the Qubit dsDNA BR assay The Qubit assays were designed to be performed at room temperature as temperature fluctuations can influence the accuracy of the assay Qubit dsDNA BR Assay Kits 6 How the Qubit 2 0 Fluorometer Calculates Concentration 120 Standards Raw Data Curve Fitting Algorithm Q L oO 1 Relative fluorescence gt O O oO to oO L 0 1 2 3 4 5 6 Concentration of dsDNA g mL Figure 3 The curve fitting algorithm used to determine concentration in the Qubit dsDNA BR assay The Qubit 2 0 Fluorometer generates concentration data based on the relationship between the two standards used in calibration This plot shows the line corresponding to the curve fitting algorithm a modified Hill plot used in the calculation of concentration data for the Qubit dsDNA BR assay For reference the positions of the standards and a set of data points from an actual experiment are shown superimposed onto the line demonstrating that the curve fitting algorithm gives accurate values for quantitation Contaminants Tolerated by the Qubit dsDNA BR Assay Table 2 Effect of contaminants in the Qubit dsDNA BR assay tested over the range 0 01 ug mL to 5 ug mL Contaminant Final Concentration Concentration in C
108. you stop scrolling the Qubit 2 0 Fluorometer calculates the original sample concentration based on the measured assay concentration To change the units in which the original sample concentration is displayed press ng mL A pop up window showing the current unit selection indicated by an adjacent red dash opens Select the unit for your original sample concentration by touching the desired unit in the unit selection pop up window To close the unit selection pop up window touch anywhere on the screen outside the pop up The Qubit 2 0 Fluorometer automatically converts the units to your selection once the unit selection pop up window is closed Note The unit button next to your sample concentration reflects the change in the units e g if you change the unit to pg L the button will display pg L To save the data from your calculation to the Qubit 2 0 Fluorometer press Save on the Dilution Calculator screen The last calculated value of your measurement will be saved as a CSV file and tagged with a time and date stamp To exit the Dilution Calculator screen press any navigator button on the bottom of the screen or Read Next Sample Note When you navigate away from the Dilution Calculator screen the Qubit 2 0 Fluorometer saves the last values for the sample volume and the units in the Dilution Calculator screen only Returning to the Dilution Calculator screen displays these last selected values BSA Ovalb

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