EXPRESS SYBR GreenER qPCR SuperMixes and Two
Contents
1. StepOnePlus 0 4 ul 50X 500 nM and PRISM 7000 and 7700 AB 7500 Stratagene Mx3000P Mx3005P and 0 04 ul 500X 50 nM Mx4000 Continued on next page 11 Universal Kits continued Fluorescein for Bio Rad iCycler Instruments 12 Bio Rad iCycler instruments require the collection of well factors before each run to compensate for any instrument or pipetting non uniformity Well factors for SYBR GreenER experiments are calculated using an additional fluorophore fluorescein Well factors are collected using either a separate plate containing fluorescein in each well External Well Factors or the experimental plate with fluorescein spiked into the qPCR master mix Dynamic Well Factors You must select the method when you start each run using the iCycler Fluorescein is available separately from Bio Rad or Fluorescein NIST Traceable Standard is available from Invitrogen as a 50 uM solution see page 20 for ordering information External Well Factors The Bio Rad iCycler instruction manual provides instructions on preparing and using the External Well Factor plate The iCycler will automatically insert a 3 cycle program before your experimental cycling program to perform the External Well Factor reading Note The iCycler iQ5 and MyiQ systems allow you to save the data from an External Well Factor reading as a separate file which can then be referenced for future readings2. Select the Persistent Well Factor setting when you are entering the cycling program to reference this saved file Dynamic Well Factors For Dynamic Well Factor readings the user must add fluorescein to the qPCR master mix at a final concentration of 10 20 nM Consult your Bio Rad iCycler instruction manual for details Note that if you select the Dynamic Well Factor option the instrument will automatically insert a 90 second incubation at 95 C before the initial 95 C denaturation step Continued on next page Universal Kits continued General Cycling Programs The following cycling programs have been developed as a general starting point when using EXPRESS SYBR GreenER qPCR SuperMix Universal The fast cycling program was developed using the AB 7500 in Fast mode Note This mix is highly robust and can be used with a wide range of cycling programs on different instruments If you have an alternative program that you want to use test it with this mix Fast Cycling Program developed using the AB 7500 in Fast mode 95 C for 20 seconds 40 cycles of 95 C for 3 seconds 60 C for 30 seconds Optional Melting curve analysis 60 C 95 C refer to instrument manual for specific programming Standard Cycling Program 50 C for 2 minutes UDG incubation 95 C for 2 minutes 40 cycles of 95 C for 15 seconds 60 C for 1 minute Optional Melting curve analysis 60 C 95 C refer to instrument manual for sp
3. are at the bottom of the tube plate centrifuge briefly if needed 4 Place reactions in a real time instrument programmed as described on the previous pages Collect data and analyze results 5 Optional The specificity of the PCR products can be checked by agarose gel electrophoresis 15 Kits with Premixed ROX Guidelines and Protocols Introduction Additional Materials Required Premixed ROX Concentration This section provides guidelines and protocols for using EXPRESS SYBR GreenER qPCR SuperMix with Premixed ROX The following items are supplied by the user e DNA starting material e DEPC treated water e Gene specific primers e Microcentrifuge e Thermal cycler e PCR tubes plates ROX Reference Dye is included in the SuperMix at a final concentration of 500 nM which is compatible with Applied Biosystems 7900HT 7300 StepOne StepOnePlus GeneAmp 5700 and PRISM 7000 and 7700 Cycling The following general cycling programs have been Programs developed as a starting point when using EXPRESS SYBR GreenER qPCR SuperMix with Premixed ROX on various instruments The fast cycling program is designed for the AB 7900HT and StepOne Note This mix is highly robust and can be used with a wide range of cycling programs on different instruments If you have an alternative program that you want to use test it with this mix Fast Cycling Program developed Standard Cycling Program using the AB 790
4. 0 pl 1 Fora single reaction combine the following components in a tube on ice For multiple reactions prepare a master mix without RNA 5X VILO Reaction Mix 4ul 10X SuperScript Enzyme Mix 2 ul RNA up to 2 5 ug x pl DEPC treated water to 20 ul 2 Gently mix tube contents and incubate at 25 C for 10 minutes 3 Incubate tube at 42 C for 60 minutes 4 Terminate the reaction at 85 C at 5 minutes 5 Use diluted or undiluted cDNA in qPCR see the guidelines for cDNA use in qPCR on page 9 Alternatively store the cDNA at 20 C until use General qPCR Guidelines and Parameters qPCR Setup and Conditions cDNA Genomic or Plasmid DNA Starting material for qPCR can be cDNA genomic DNA or plasmid DNA Maintain a sterile environment when handling DNA to avoid any contamination from DNases Make sure all equipment that comes in contact with DNA is sterile including pipette tips and microcentrifuge tubes qPCR reaction volumes can be scaled from 5 pl to 100 ul depending on the instrument For instrument specific guidelines see the section for each type of SuperMix If you are using cDNA as starting material If you started with lt 100 ng of total RNA up to 10 of the qPCR reaction volume may be undiluted cDNA e g for a 20 11 qPCR use up to 2 pl of undiluted cDNA If you started with gt 100 ng total RNA we recommend diluting the cDNA prior to qPCR because higher concentrations of cDNA will affec
5. 0HT and StepOne 50 C for 2 minutes UDG incubation 95 C for 20 seconds 95 C for 2 minutes 40 cycles of 40 cycles of 95 C for 1 second 95 C for 15 seconds 60 C for 20 seconds 60 C for 1 minute Optional Melting curve analysis Optional Melting curve analysis 60 C 95 C refer to instrument 60 C 95 C refer to instrument manual for specific programming manual for specific programming 16 Continued on next page Kits with Premixed ROX continued 384 Well Plate Volumes qPCR Protocol For 384 well plates we recommend a maximum reaction volume of 10 yl per well Use the protocol below as a general starting point for qPCR with EXPRESS SYBR GreenER qPCR SuperMix with Premixed ROX Scale the reaction volume as needed for your real time instrument 1 Set up reactions on ice Volumes for a 20 11 reaction size are provided component volumes can be scaled as needed For 384 well plates we recommend a maximum reaction volume of 10 pl per well Always prepare a master mix of common components for multiple reactions 20 ul rxn EXPRESS SYBR GreenER qPCR SuperMix with Premixed ROX 10 pl 10 uM forward primer 200 nM final 0 4 ul 10 pM reverse primer 200 nM final 0 4 ul Template DNA see page 9 Xul DEPC treated water to 20 pl 2 Prepare no template control NTC reactions to test for DNA contamination of the enzyme primer mixes 3 Cap or seal each PCR tube plate and gently mix Mak
6. CR cycling providing an automatic hot start in qPCR for increased sensitivity specificity and yield Continued on next page Overview continued Heat labile Uracil DNA Glycosylase UDG ROX Reference Dye Additional Materials Required Heat labile UDG and dUTP in the qPCR SuperMix prevent the reamplification of carryover PCR products between reactions Lindahl et al 1977 Longo et al 1990 dUTP ensures that any amplified DNA will contain uracil while heat labile UDG removes uracil residues from single or double stranded DNA The heat labile form of UDG used in this kit is completely inactivated at temperatures of 50 C and higher and will not degrade amplicons following qPCR thus enabling their use for downstream applications such as cloning ROX Reference Dye is either premixed in the qPCR SuperMix or included as a separate component to normalize the fluorescent signal between reactions for instruments that are compatible with this option The following items are supplied by the user e Template RNA Two Step Kits or DNA qPCR SuperMixes only e Gene specific primers e DEPC treated water e Microcentrifuge e Thermal cycler e Optional Normalization dye for instruments that do not use ROX e PCR tubes plates Instrument Compatibility Universal SuperMix Kits with Premixed ROX EXPRESS SYBR GreenER qPCR SuperMix Universal includes ROX Reference Dye as a separate tube and can be used wit
7. agents dilutions volumes and cycling parameters have been used Template contains inhibitors nucleases or proteases or has otherwise been degraded Purify or re purify your template Primer design is suboptimal Verify your primer selection We recommend using validated pre designed primers or design primers using dedicated software programs or primer databases 18 Continued on next page Troubleshooting continued Problem Cause Solution PCR product is qPCR instrument Confirm that you are using the evident on a gel but settings are correct instrument settings dye not in the qPCR incorrect selection reference dye filters and graph acquisition points Problems with your See your instrument manual for tips specific qPCR and troubleshooting instrument PCR efficiency is Template contains Purify or re purify your template above 110 inhibitors nucleases or proteases or has otherwise been degraded Too much sample added to reactions Inhibitors in the template may result in changes in PCR efficiency between dilutions Decrease the concentration of cDNA see the guidelines for cDNA concentration on page 9 Nonspecific products may be amplified Use melting curve analysis if possible and or run the PCR products on a 4 agarose gel after the reaction to identify contaminants Suboptimal primer design may lead to nonspecific products Use validated pre d
8. and can be used with very low and very high amounts of input RNA up to 2 5 ug total RNA in a 20 pl reaction giving a linear response in message abundance as measured by qPCR The 10X SuperScript Enzyme Mix includes SuperScript II RT RNaseOUT Recombinant Ribonuclease Inhibitor and a proprietary helper protein The 5X VILO Reaction Mix includes random primers MgCh and dNTPs in a buffer formulation that has been optimized for qRT PCR e Starting material using the VILO kit can range up to 2 5 ug total RNA in a 20 1 cDNA synthesis reaction Note that for qPCR using SYBR GreenER SuperMixes you will need to dilute the cDNA generated from total RNA quantities above 100 ng e To isolate total RNA we recommend the PureLink Micro to Midi Total RNA Purification System TRIzol Reagent or the PureLink 96 Total RNA Purification Kit see page 20 Isolation of mRNA is typically not necessary although incorporating this step may improve the yield of specific cDNAs e High quality intact RNA is essential for accurate quantification in qRT PCR e DNase I Amplification Grade may be used to eliminate genomic DNA contamination from the total RNA see page 20 Continued on next page 5 First Strand cDNA Synthesis continued General Handling of RNA Determining Total RNA Quality When working with RNA e Use proper microbiological aseptic technique e Wear latex gloves while handling reagents mate
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10. andards for a standard curve The assay is performed in a microtiter plate and can be read using a standard fluorescent microplate reader UV Absorbance 1 Dilute an aliquot of the total RNA sample in 10 mM Tris HCl pH 7 5 Mix well Transfer to a cuvette 1 cm path length Note The RNA must be in a neutral pH buffer to accurately measure the UV absorbance 2 Determine the OD2 60 of the solution using a spectrophotometer blanked against 10 mM Tris HCl pH 7 5 Calculate the amount of total RNA using the following formula Total RNA ug OD2 x 40 ug 1 OD260 x 1 ml x dilution factor x total sample volume ml Example Total RNA was eluted in water in a total volume of 150 pl A 40 l aliquot of the eluate was diluted to 500 ul in 10 mM Tris HCl pH 7 5 An OD260 of 0 188 was obtained The amount of RNA in the sample is Total RNA ug 0 188 x 40 pg 1 OD260 x 1 ml x 12 5 x 0 15 14 1 pg Continued on next page First Strand cDNA Synthesis continued Guidelines for e Shorter incubation times and or higher temperatures cDNA may be used e g 50 C for 30 minutes but may result Synthesis in reduced yields of cDNA e For increased yields of cDNA longer incubation times may be used up to 120 minutes at 42 C cDNA The following protocol has been optimized for generating Synthesis first strand cDNA using the SuperScript VILO cDNA Protocol Synthesis Kit The reaction volume may be scaled as needed up to 10
11. ats and are compatible with both rapid and standard qPCR cycling conditions All EXPRESS SYBR GreenER qPCR SuperMixes include Platinum Tag DNA polymerase SYBR GreenER fluorescent dye MgCl heat labile uracil DNA glycosylase UDG dNTPs with dUTP instead of dTTP and stabilizers SuperMix with Premixed ROX The qPCR SuperMix with premixed ROX includes ROX Reference Dye at a final concentration of 500 nM to normalize the fluorescent signal on instruments that are compatible with this option Universal SuperMix The Universal SuperMix includes ROX as a separate component for instruments that use ROX at a different concentration or do not require ROX Two Step qRT PCR Kits These kits include a qPCR SuperMix and the SuperScript VILO cDNA Synthesis Kit for cDNA synthesis prior to qPCR The VILO kit provides enhanced cDNA synthesis efficiency and can be used with very low and very high amounts of input RNA from 1 pg up to 2 5 ug total RNA in a 20 1 reaction giving a linear response in message abundance as measured by qPCR Continued on next page Overview continued Advantages of the Kits SYBR GreenER Fluorescent Dye Platinum Taq DNA Polymerase Highly robust qPCR SuperMix can accommodate a wide range of cycling conditions and reaction volumes and combines highly sensitive detection with a broad quantification range e SYBR GreenER dye in these formulations provides higher sensitivity a
12. centration of 200 nM per primer is effective for most reactions Optimal results may require a titration of primer concentrations between 100 and 500 nM Melting curve analysis should always be performed following real time qPCR to identify the presence of primer dimers and analyze the specificity of the reaction Program your instrument for melting curve analysis using the instructions provided with your specific instrument Universal Kits Guidelines and Protocols Introduction This section provides guidelines and protocols for using EXPRESS SYBR GreenER qPCR SuperMix Universal Additional The following items are supplied by the user Materials e DNA starting material Required DEPC treated water Gene specific primers Microcentrifuge Thermal cycler PCR tubes plates ROX ROX Reference Dye is supplied as a separate tube in the Reference Dye Universal Kits ROX is recommended for fluorescence Concentration normalization on Applied Biosystems instruments and is optional for Stratagene and Eppendorf instruments It is not required on other instruments ROX is composed of a glycine conjugate of 5 carboxy X rhodamine succinimidyl ester and is supplied at a concentration of 25 uM Use the following table to determine the amount of 25 uM ROX to use with a particular instrument Amount of Effective Fold Final ROX Instrument ROX per 20 y1 Concentration of Concentriton reaction 25 uM ROX AB 7300 7900HT StepOne
13. components and Storage in each kit are listed below Storage Store all components at 20 C for long term storage EXPRESS qPCR SuperMixes may be stored at 4 8 C for up to one month EXPRESS SYBR GreenER qPCR Supermix 11784 200 11784 01K Universal EXPRESS SYBR GreenER qPCR SuperMix Universal ROX Reference Dye 500 ul 5 x 500 pl EXPRESS SYBR GreenER qPCR Supermix with 11794 200 11794 01K Premixed ROX EXPRESS SYBR GreenER qPCR SuperMix with 5 ml 5x5ml Premixed ROX EXPRESS Two Step SYBR GreenER Universal 11782 200 11782 01K EXPRESS SYBR GreenER qPCR SuperMix 5 ml 5x5ml Universal ROX Reference Dye 900pl 5x 500 ul SuperScript VILO cDNA Synthesis Kit 50 rxns 5X VILO Reaction Mix 20 ul each 20 ul each 10X SuperScript Enzyme Mix EXPRESS Two Step SYBR GreenER with 11792 200 Premixed ROX ERN EXPRESS SYBR GreenER qPCR SuperMix with Premixed ROX SuperScript VILO cDNA Synthesis Kit 50 rxns 250 rxns 5X VILO Reaction Mix 20 ul each 20 ul each 10X SuperScript Enzyme Mix Intended Use For research use only Not intended for human or animal diagnostic or therapeutic uses iv Overview Introduction EXPRESS SYBR GreenER qPCR SuperMixes and Two Step qRT PCR Kits provide components for real time quantitative PCR qPCR and two step reverse transcription qPCR qRT PCR Components are provided in convenient SuperMix form
14. e sure that all components are at the bottom of the tube plate centrifuge briefly if needed 4 Place reactions in a real time instrument programmed as described on the previous page Collect data and analyze results 5 Optional The specificity of the PCR products can be checked by agarose gel electrophoresis 17 Troubleshooting Problem Cause Solution Signals are present in no template controls and or multiple peaks are present in the melting curve graph Template or reagents are contaminated by nucleic acids DNA cDNA Use melting curve analysis and or run the PCR products on a 4 agarose gel after the reaction to identify contaminants Take standard precautions to avoid contamination when preparing your PCR reactions Ideally amplification reactions should be assembled in a DNA free environment We recommend using aerosol resistant barrier tips Primer dimers or other primer artifacts are present Use melting curve analysis to identify primer dimers We recommend using validated pre designed primer sets or design primers using dedicated software programs or primer databases Primer contamination or truncated or degraded primers can lead to artifacts Check the purity of your primers by gel electrophoresis No PCR product is evident either in the qPCR graph or ona gel The protocol was not followed correctly Verify that all steps have been followed and the correct re
15. ecific programming Continued on next page 13 Universal Kits continued Roche The following cycling program is specific for the Roche Lig htCycler LightCycler 480 with a 96 well or 384 well plate when 480 Cycling using EXPRESS SYBR GreenER qPCR SuperMix Program Universal For detailed programming instructions consult the instrument manual Program Name Cycles Analysis Mode Pre incubation 1 None Amplification 40 45 Quantification Melting Curve 1 Melting Curves Cooling 1 None Target C Acquisition Mode ee 9 Bam Rate CCS Pre incubation 95 None 00 05 00 4 4 or 2 0 4 8 Amplification 95 None 00 00 10 4 4 or 2 0 4 8 Primer Tm None 00 00 05 2 2 2 5 minus 5 C 00 00 20 72 Single 00 00 05 4 4 or 2 0 4 8 00 00 20 Melting Curve 95 None 00 00 05 2 0 2 0 65 None 00 01 00 2 0 2 0 97 Continuous 5 10 acquisitions per C Cooling 40 None 00 00 10 2 0 2 0 greater A ramp rate of 2 0 C s is recommended for reaction volumes of 50 ul or The annealing temperature will vary depending on the melting temperature Tm of the primers Use primer Tm minus 5 C as a general starting point 3 Longer annealing and extension times may result in greater precision in target quantification 14 Continued on next page Universal Kits continued 384 Well Plate Volumes qPCR Protocol For 384 wel
16. esigned primers or design primers using dedicated software programs or primer databases PCR efficiency is below 90 The PCR conditions are suboptimal Verify that the reagents you are using have not been freeze thawed multiple times and have not remained at room temperature for too long Verify that the amount of primers you are using is correct 19 Appendix Additional Products Additional Related products are available separately from Invitrogen Products Ordering information is provided below For more information visit our website at www invitrogen com or contact Technical Service page 20 SuperScript VILO cDNA Synthesis Kit 50 rxns 11754 050 250 rxns 11754 250 10328 011 100 ml 15596 026 200 ml 15596 018 TM RNase Away Reagent DNase I Amplification Grade PureLink Micro to Midi Total RNA Purification System TRIzol Reagent PureLink Genomic DNA Mini Kit Quant iT RNA Assay Kit Quant iT DNA Assay Kit High Sensitivity Quant iT DNA Assay Kit Broad Range Fluorescein NIST Traceable Standard 50 uM Custom Primers 20 50 preps K1820 01 250 preps K1820 02 Qasr 1000 assays Q33120 1000 assays Q33130 visit www invitrogen com oligos Technical Support On the Web Visit the Invitrogen website at www invitrogen com for e Complete technical support contact information e Technical resources including manuals vector maps
17. fication continued Limited Use Label License No 274 5 Nuclease Process Trademarks of Other Companies 24 A license to perform the 5 nuclease process for research requires the use of a Licensed 5 Nuclease Kit containing Licensed Probe or the combination of an Authorized Core Kit plus Licensed Probe or license rights that may be purchased from Applied Biosystems This product is an Authorized Core Kit without Licensed Probe Its purchase price includes a limited non transferable immunity from suit under U S Patents and corresponding patent claims outside the United States owned by Roche Molecular Systems Inc or F Hoffmann La Roche Ltd Roche for using only this amount of the product in the practice of the 5 nuclease process solely for the purchaser s own internal research and development activities This product is also an Authorized Core Kit for use with service sublicenses available from Applied Biosystems This product conveys no rights under U S Patents Nos 5 804 375 6 214 979 5 538 848 5 723 591 5 876 930 6 030 787 or 6 258 569 or corresponding patent claims outside the United States expressly by implication or by estoppel No right under any other patent claims such as apparatus or system claims and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is hereby granted express
18. h a wide range of real time instruments including the following e Applied Biosystems 7900HT 7300 7500 StepOne StepOnePlus GeneAmp 5700 and PRISM 7000 and 7700 e Bio Rad MJ Research iCycler iQ i105 and MyiQ DNA Engine Opticon and Opticon 2 and Chromo4 Real Time Detector e Cepheid Smart Cycler e Corbett Research Rotor Gene 3000 e Eppendorf Mastercycler ep realplex e Roche LightCycler 480 e Stratagene Mx3000P Mx3005P and Mx4000 EXPRESS SYBR GreenER qPCR SuperMix with Premixed ROX can be used with real time instruments that are compatible with ROX Reference Dye at a final concentration of 500 nM These include the following Applied Biosystems instruments e 7900HT e 7300 e StepOne e StepOnePlus e GeneAmp 5700 e PRISM 7000 and 7700 Methods First Strand cDNA Synthesis Introduction SuperScript VILO cDNA Synthesis Kit Input RNA This section provides guidelines and a protocol for first strand cDNA synthesis from RNA using the SuperScript VILO cDNA Synthesis Kit which is included with the two step kits and is also available separately If you are performing qPCR using DNA from another source you can skip this section This cDNA synthesis kit is included with the EXPRESS Two Step SYBR GreenER qRT PCR Kits and is also available separately see page 20 for ordering information It provides enhanced cDNA synthesis efficiency
19. invitrogen by technologies EXPRESS SYBR GreenER qPCR SuperMixes and Two Step qRT PCR Kits Catalog nos 11782 200 01K 11784 200 01K 11792 200 01K and 11794 200 01K Rev Date 2 June 2010 Manual part no A10328 MAN0000690 ii Table of Contents Kit Contents and Storage c ccccccccceseesesssseneesesssesesceceseneeesesssnsneneness iv CNV CEVA CW pda niia aiaa di chs bee ce ab edk db A A a toess 1 Instrument Compatibility 00 0 cece ceneneeseecseeeeceseneneteesesesnanenenes 4 Method S decease enina kaina otenntsacs secsunts cdnsaneasnbawassavashecmesabnceathccedenauiausste 5 First Strand cDNA Synthesis cccccccccsesesnsneseseseeeeceeenenenenesesseneenenes 5 General qPCR Guidelines and Parameters cccccccceseeseseeeeeetetenes 9 Universal Kits Guidelines and Protocols cccccee eee eects teens 11 Kits with Premixed ROX Guidelines and Protocols eee 16 Troubleshooting eenah ea aaea eE EREE AERE 18 Appendix cies cesceders sais srerseodwennd sevetrenatanscenesadnavavaneceduecnraraveananeunddas 20 Additional Products sau1 ass soe opal Sas EO eae ee 20 Technical Support sensio neea os tone takin a rear nee eros 21 Purchaser Notification cecsesscssssecsceseeeceeeeesececeaesececeaeseceeseceeeasseeeees 23 Kelerenn a aun se ean phate nena 25 iii Kit Contents and Storage Kit EXPRESS SYBR GreenER qPCR SuperMixes and Two Components Step qRT PCR Kits are shipped on dry ice The
20. l plates we recommend a maximum reaction volume of 10 yl per well Use the protocol below as a general starting point for qPCR with EXPRESS SYBR GreenER qPCR SuperMix Universal Scale the reaction volume as needed for your real time instrument ROX is recommended for Applied Biosystems instruments and optional for Stratagene and Eppendorf instruments see page 11 Bio Rad iCycler instruments use fluorescein instead of ROX for Dynamic Well Factor readings see page 12 1 Set up reactions on ice Volumes for a 20 11 reaction size are provided component volumes can be scaled as needed For 384 well plates we recommend a maximum reaction volume of 10 pl per well Always prepare a master mix of common components for multiple reactions 20 ul rxn EXPRESS SYBR GreenER qPCR SuperMix Universal 10 ul 10 uM forward primer 200 nM final 0 4 ul 10 pM reverse primer 200 nM final 0 4 pl ROX Reference Dye 25 uM 0 4 pl 0 04 ul Template DNA see page 9 X pl DEPC treated water to 20 pl Consult instrument documentation The iCycler uses fluorescein instead of ROX for Dynamic Well Factor readings 10 20 nM final concentration see page 12 See the table on page 11 for the amount concentration of ROX to use for your specific instrument 2 Prepare no template control NTC reactions to test for DNA contamination of the enzyme primer mixes 3 Cap or seal each PCR tube plate and gently mix Make sure that all components
21. labile switches high temperature triggering for the polymerase chain reaction Biotechnology 12 506 509 Wittwer C T Herrmann M G Moss A A and Rasmussen R P 1997 Continuous fluorescence monitoring of rapid cycle DNA amplification BioTechniques 22 130 138 2010 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners 25 Notes invitrogen by Life technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
22. ly by implication or by estoppel This product is for research purposes only Diagnostic uses require a separate license from Roche Further information regarding the 5 nuclease licensing program may be obtained from the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA iCycler MyIQ Mx3000P Mx3005 Mx4000 Rotor Gene DNA Engine Opticon Chromo 4 Smart Cycler LightCycler Mastercyler are trademarks or registered trademarks of their respective companies References Chou Q Russell M Birch D Raymond J and Bloch W 1992 Prevention of pre PCR mis priming and primer dimerization improves low copy number amplifications Nucl Acids Res 20 1717 1723 Ishiguro T Saitoh J Yawata H Yamagishi H Iwasaki S and Mitoma Y 1995 Homogeneous quantitative assay of hepatitis C virus RNA by polymerase chain reaction in the presence of a fluorescent intercalater Anal Biochem 229 207 Lindahl T Ljungquist S Siegert W Nyberg B and Sperens B 1977 DNA N glycosidases properties of uracil DNA glycosidase from Escherichia coli J Biol Chem 252 3286 3294 Longo M Berninger M and Hartley J 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 Sharkey D J Scalice E R Christy K G Atwood S M and Daiss J L 1994 Antibodies as thermo
23. nd lower PCR inhibition than other fluorescent double stranded DNA binding dyes e Platinum Taq DNA Polymerase provides an automatic antibody mediated hot start in PCR for increased sensitivity specificity and yield and has a short activation time for the rapid cycling of fast qPCR instruments e Heat labile UDG and dUTP in the SuperMix prevent amplification of carryover PCR products between reactions and the heat labile form of the enzyme is completely inactivated during normal qPCR cycling eliminating any downstream degradation of amplicons e SuperScript VILO cDNA Synthesis Kit included in the two step kits provides high yields of cDNA and linear output over a very broad range of RNA input quantities SYBR GreenER fluorescent dye is a double stranded DNA dsDNA binding dye that in this formulation provides higher sensitivity and lower PCR inhibition than SYBR Green I dye It can be used on real time PCR instruments calibrated for SYBR Green I dye without any change of filters or settings In qPCR as dsDNA accumulates SYBR GreenER dye generates a signal that is proportional to the DNA concentration Ishiguro et al 1995 Wittwer et al 1997 Platinum Tag DNA Polymerase is recombinant Taq DNA polymerase complexed with proprietary antibodies that block polymerase activity at ambient temperatures Chou et al 1992 Sharkey et al 1994 Activity is restored after the initial denaturation step in P
24. r 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 For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product 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 Life Technologies is willing to accept return of the product with a full refund For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 or e mail outlicensing lifetech com Licensed to Life Technologies Corporation under U S Patent Nos 5 338 671 5 587 287 and foreign equivalents for use in research only Continued on next page 23 Purchaser Noti
25. rials and RNA samples to prevent RNase contamination e Use disposable individually wrapped sterile plasticware for all procedures e Use aerosol resistant pipette tips e Dedicate a separate set of pipettes buffers and enzymes for RNA work e Use RNase free microcentrifuge tubes To decontaminate untreated tubes soak overnight in a 0 01 v v aqueous solution of diethylpyrocarbonate DEPC rinse with sterile distilled water and autoclave RNase Away Reagent a non toxic solution available from Invitrogen can be used to remove RNase contamination from surfaces Total RNA quality can be analyzed using a bioanalyzer such as the Agilent 2100 bioanalyzer with an RNA LabChip Alternatively total RNA can be analyzed by agarose gel electrophoresis RNA isolated using the PureLink kits or TRIzol Reagent typically has a 28S to 18S band ratio of gt 1 5 RNA is judged to be intact if discreet 28S and 18S ribosomal RNA bands are observed Continued on next page First Strand cDNA Synthesis continued Determining Total RNA Yield Total RNA can be quantitated using the Quant iT RNA Assay Kit or UV absorbance at 260 nm Quant iT RNA Assay Kit The Quant iI RNA Assay Kit provides a rapid sensitive and specific method for RNA quantitation with minimal interference from DNA protein or other common contaminants that affect UV absorbance readings The kit contains a quantitation reagent and pre diluted st
26. s to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service please contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to the specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether e
27. t the signal baseline in SYBR GreenER SuperMix reactions For example if you started with 2 ug of total RNA prepare a 20 fold dilution of the resulting cDNA to achieve the concentration equivalent of starting with 100 ng of RNA Then use up to 2 pl of the diluted cDNA in a 20 ul qPCR lt 10 of qPCR volume Note that detecting high abundance genes in undiluted cDNA may result in very low CTs in qPCR leading to reduced quantification accuracy Prepare a dilution series of the cDNA template for the most accurate results If you are using genomic or plasmid DNA as starting material Use up to 100 ng of genomic DNA or 10 10 copies of plasmid DNA in a 10 11 volume Note that 1 ug of plasmid DNA contains 9 1 x 10 copies divided by the plasmid size in kilobases Continued on next page 9 General qPCR Guidelines and Parameters continued Primer Specifications Melting Curve Analysis 10 Primer design is one of the most important parameters when using EXPRESS SYBR GreenER qPCR SuperMixes We strongly recommend using a primer design software program such as OligoPerfect available on the Web at www invitrogen com oligos or Vector NTI In addition to designing primers for optimal efficiency programs such as these will automatically perform a BLAST search of NCBI databases to ensure that primers are target specific When designing primers the amplicon length should be approximately 80 250 bp A final con
28. xpressed or implied including any warranty of merchantability or fitness for a particular purpose Purchaser Notification Limited Use Label License No 5 Invitrogen Technology Limited Use Label License No 14 Direct Inhibition by Anti Polymerase Antibodies 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 not to 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 o
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