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AllPrep® DNA/RNA/Protein Mini Handbook

1. 16 AllPrep DNA RNA Protein Mini Handbook 09 2011 Effect of homogenization on DNA yield and integrity The yield and integrity of genomic DNA purified using the AllPrep DNA RNA Protein Mini Kit depends on the method used for disruption and homogenization Homogenization with the TissueRuptor or other rotor stator homogenizer or the TissueLyser or other bead mill results in higher DNA yields but also in greater DNA fragmentation depending on the homogenization time and intensity In contrast gentler homogenization with the QlAshredder or a syringe and needle allows purification of longer DNA fragments However as longer DNA fragments are more difficult to elute DNA yields may be lower AllPrep DNA RNA Protein Mini Handbook 09 2011 17 Protocol Simultaneous Purification of Genomic DNA Total RNA and Total Protein from Animal and Human Cells Determining the correct amount of starting material It is essential to use the correct amount of starting material in order to obtain optimal nucleic acid yield and purity The minimum amount is generally 100 cells while the maximum amount depends on E The RNA content of the cell type M The DNA binding capacity of the AllPrep DNA spin column E The RNA binding capacity of the RNeasy spin column 100 pg RNA m The volume of Buffer RLT required for efficient lysis the maximum volume of Buffer RLT that can be used limits the maximum amount of starting material to 1 x 10 cel
2. Note Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the AllPreo DNA and RNeasy spin columns Homogenization with the TissueRuptor or Tissuelyser generally results in higher nucleic acid yields than with other methods However prolonged homogenization with these homogenizers results in greater DNA fragmentation Table 7 Volumes of Buffer RLT for tissue disruption and homogenization Amount of starting material Volume of Buffer RLT lt 20 mg 350 pl or 600 pl 20 30 mg 600 pl Use 600 pl Buffer RLT for stabilized tissues or for difficult to lyse tissues AllPrep DNA RNA Protein Mini Handbook 09 2011 29 3a Disruption and homogenization using the TissueRuptor Place the tissue in a suitably sized vessel Add the appropriate volume of Buffer RLT see Table 7 Note Use a suitably sized vessel with sufficient extra headspace to accommodate foaming which may occur during homogenizaation Generally round bottomed tubes allow more efficient disruption and homogenization than conical bottomed tubes Place the tip of the disposable probe into the vessel and operate the TissueRuptor at full speed until the lysate is homogeneous usually 30 s Proceed to step 4 Note To avoid damage to the TissueRuptor and disposable probe during operation make sure the tip of the probe remains submerged in the buffer Foaming may occur during homogenization If this happens let th
3. Glassware Glassware should be treated before use to ensure that it is RNase free Glassware used for RNA work should be cleaned with a detergent thoroughly rinsed and oven baked at 240 C for at least 4 hours overnight if more convenient before use Autoclaving alone will not fully inactivate many RNases Alternatively glassware can be treated with DEPC diethyl pyrocarbonate Fill glassware with 0 1 DEPC 0 1 in water allow to stand overnight 12 hours at 37 C and then autoclave or heat to 100 C for 15 minutes to eliminate residual DEPC Electrophoresis tanks Electrophoresis tanks should be cleaned with detergent solution e g 0 5 SDS thoroughly rinsed with RNase free water and then rinsed with ethanol and allowed to dry Solutions Solutions water and other solutions should be treated with 0 1 DEPC DEPC is a strong but not absolute inhibitor of RNases It is commonly used at a concentration of 0 1 to inactivate RNases on glass or plasticware or to create RNase free solutions and water DEPC inactivates RNases by covalent modification Add 0 1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution Let the solution incubate for 12 hours at 37 C Autoclave for 15 minutes to remove any trace of DEPC DEPC will react with primary amines and cannot be used directly to treat Tris buffers DEPC is highly unstable in the presence of Tris buffers and decomposes rapidl
4. see Spectrophotometric quantification of RNA below For small amounts of RNA however it may be difficult to determine amounts photometrically Small amounts of RNA can be accurately quantified using an Agilent 2100 bioanalyzer quantitative RT PCR or fluorometric quantification Spectrophotometric quantification of RNA To ensure significance Azs readings should be greater than 0 15 An absorbance of 1 unit at 260 nm corresponds to 44 pg of RNA per ml Ayo 1 gt 44 pg ml This relation is valid only for measurements at a neutral pH Therefore if it is necessary to dilute the RNA sample this should be done in a buffer with neutral pH As discussed below see Purity of RNA page 45 the ratio between the absorbance values at 260 and 280 nm gives an estimate of RNA purity When measuring RNA samples be certain that cuvettes are RNase free especially if the RNA is to be recovered after spectrophotometry This can be accomplished by washing cuvettes with 0 1 M NaOH 1 mM EDTA followed by washing with RNase free water see Solutions page 43 Use the buffer in which the RNA is diluted to zero the spectrophotometer An example of the calculation involved in RNA quantification is shown below Volume of RNA sample 100 pl Dilution 10 pl of RNA sample 490 pl of 10 mM Tris Cl pH 7 0 1 50 dilution Measure absorbance of diluted sample in a 1 ml cuvette RNase free A 0 2 When working with chemicals al
5. w v SDS or 8 M urea or increase the volume of resuspension buffer Protein bands on SDS PAGE gel or western blot show a zig zag pattern or smear a Protein sample contains Insoluble material may influence the running insoluble material behavior of the gel Repeat steps 19 and 20 of the protein precipitation procedure making sure that no insoluble material is transferred to your downstream application b Protein shows no clear pattern The quality of SDS PAGE can be influenced in SDS PAGE by several parameters independent of protein quality Vary the protein load and or the polyacrylamide concentration of the gel which should be according to to molecular mass of the protein of interest Incubation of the sample for 10 min at 46 C before loading instead of 95 C can improve the resolution AllPrep DNA RNA Protein Mini Handbook 09 2011 41 Appendix A General Remarks on Handling RNA Handling RNA Ribonucleases RNases are very stable and active enzymes that generally do not require cofactors to function Since RNases are difficult to inactivate and even minute amounts are sufficient to destroy RNA do not use any plasticware or glassware without first eliminating possible RNase contamination Great care should be taken to avoid inadvertently introducing RNases into the RNA sample during or after the purification procedure In order to create and maintain an RNase free environment the following precautions must be taken durin
6. For greatest accuracy readings should be between 0 1 and 1 0 Using a standard 1 cm path length an absorbance of 1 unit at 260 nm corresponds to 50 pg genomic DNA per ml Aygo 1 gt 50 pg ml This relation is valid only for measurements made at neutral pH Therefore samples should be diluted in a low salt buffer with neutral pH e g Tris Cl pH 7 0 Use the buffer in which the DNA is diluted to zero the spectrophotometer An example of the calculation involved in DNA quantification is shown below Volume of DNA sample 100 pl Dilution 20 pl of DNA sample 180 pl of buffer 1 10 dilution Measure absorbance of diluted sample in a 0 2 ml cuvette Az 0 2 Concentration of DNA sample 50 pg ml x Azo x dilution factor 50 pg ml x 0 2 x 10 100 pg ml Total amount concentration x volume of sample in milliliters 100 pg ml x 0 1 ml 10 pg of DNA RNA concentration can also be determined by measuring the absorbance at 260 nm If the eluate contains both DNA and RNA a fluorometer must be used to quantify the DNA When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier AllPrep DNA RNA Protein Mini Handbook 09 2011 49 Determination of DNA purity The ratio of the readings at 260 nm and 280 nm Ag60 Azgo provides an estimate of DNA purity with respect to
7. RLT see Table 7 m Pipet the lysate directly into a QlAshredder spin column placed in a 2 ml collection tube and centrifuge for 2 min at full speed Proceed to step 4 3d Disruption using a mortar and pestle followed by homogenization using a needle and syringe m Immediately place the weighed tissue in liquid nitrogen and grind thoroughly with a mortar and pestle m Decant tissue powder and liquid nitrogen into an RNase free liquid nitrogen cooled 2 ml microcentrifuge tube not supplied Allow the liquid nitrogen to evaporate but do not allow the tissue to thaw m Add the appropriate volume of Buffer RLT see Table 7 and homogenize by passing the lysate at least 5 times through a blunt 20 gauge 0 9 mm diameter needle fitted to an RNase free syringe Proceed to step 4 Centrifuge the lysate for 3 min at full speed Carefully remove the supernatant by pipetting and transfer it to an AllPrep DNA spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 30 s at gt 8000 x g 210 000 rpm In some preparations very small amounts of insoluble material will be present after the 3 min centrifugation making the pellet invisible Note Make sure that no liquid remains on the column membrane after centrifugation If necessary repeat the centrifugation until all liquid has passed through the membrane Place the AllPrep DNA spin column in a new 2 ml collection tube supplied and store at
8. application Due to the strong denaturing conditions with Buffer RLT which is necessary to inactivate RNases and proteases the precipitated proteins may show reduced solubility Vortex for several minutes or disaggregate the pellet by pipetting up and down several times Depending on the sample type the pellet may contain proteins or other cellular components that are not soluble For details on how to solubilize these samples see Troubleshooting Guide on page 36 For easier dissolving or if the proteins need to be quantified prior to SDS PAGE dissolve the pellet in 5 w v SDS or 8 M urea For details about protein quantification see Appendix F page 53 Buffer ALO may turn yellow upon dissolving protein but this has no effect on downstream applications The buffer will turn blue again in SDS PAGE sample buffer If using a stock solution of your own sample buffer e g 5x concentration be sure to dilute it accordingly so that the protein sample to be loaded on a gel has a 1x concentration of sample buffer 19 Incubate for 5 min at 95 C to completely dissolve and denature the protein Then cool the sample to room temperature 20 Centrifuge for 1 min at full speed to pellet any residual insoluble material Use the supernatant in downstream applications such as SDS PAGE and western blotting The dissolved protein can be stored at 20 C for several months or at 4 C for several days Genomic DNA purification 21 Add
9. contaminants that absorb UV light such as protein The A260 A20 ratio is influenced considerably by pH Since water is not buffered the pH and the resulting A2s0 A20 ratio can vary greatly Lower pH results in a lower Apgo A280 ratio and reduced sensitivity to protein contamination For accurate Aygo Argo values we recommend measuring absorbance in a slightly alkaline buffer e g 10 mM Tris Cl pH 7 5 Make sure to zero the spectrophotometer with the appropriate buffer Pure DNA has an Ajgo Ago ratio of 1 7 1 9 Scanning the absorbance from 220 320 nm will show whether there are contaminants affecting absorbance at 260 nm Absorbance scans should show a peak at 260 nm and an overall smooth shape Determination of DNA length The precise length of genomic DNA can be determined by pulsed field gel electrophoresis PFGE through an agarose gel The DNA should be concentrated by alcohol precipitation and reconstituted by gentle agitation in approximately 30 pl TE buffer pH 8 0 for at least 30 minutes at 60 C Avoid drying the DNA pellet for more than 10 minutes at room temperature 15 25 C since over dried genomic DNA is very difficult to redissolve Load 3 5 pg of DNA per well Standard PFGE conditions are as follows E 1 agarose gel in 0 5x TBE electrophoresis buffert MH Switch intervals 5 40 seconds M Run time 17 hours E Voltage 170 V Wilfinger W W Mackey M and Chomezynski P 1997 Effect of pH and ionic
10. final DNA yield however may be reduced AllPrep DNA RNA Protein Mini Handbook 09 2011 35 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Comments and suggestions Clogged AllPrep DNA or RNeasy spin column a Inefficient disruption and or See Disrupting and homogenizing starting homogenization material page 14 for details on disruption and homogenization methods Increase gforce and centrifugation time if necessary In subsequent preparations reduce the amount of starting material see protocols pages 18 and 27 and or increase the homogenization time b Too much starting material Reduce the amount of starting material It is essential to use the correct amount of starting material see page 12 c Centrifugation temperature The centrifugation temperature should be too low 20 25 C Some centrifuges may cool to below 20 C even when set at 20 C This can cause formation of precipitates that can clog the spin column If this happens set the centrifugation temperature
11. length of the purified DNA usually 15 30 kb depends strongly on the homogenization conditions If longer DNA fragments are required keep the homogenization time to a minimum or use a gentler homogenization method if possible e g use a QlAshredder homogenizer instead of a rotor stator homogenizer Nucleic acid concentration too low Elution volume too high Elute nucleic acids in a smaller volume Do not use less than 50 pl Buffer EB for the AllPrep DNA spin column or less than 30 pl RNase free water for the RNeasy spin column Although eluting in smaller volumes results in increased nucleic acid concentrations yields may be reduced Nucleic acids do not perform well in downstream experiments a Salt carryover during elution Ensure that buffers are at 20 30 C Ensure that the correct buffer is used for each step of the procedure When reusing collection tubes between washing steps remove residual flow through from the rim by blotting on clean paper towels AllPrep DNA RNA Protein Mini Handbook 09 2011 39 Comments and suggestions b Ethanol carryover During the second wash with Buffer RPE be sure to centrifuge at 28000 x g 210 000 rpm for 2 min at 20 25 C to dry the RNeasy spin column membranes After centrifugation carefully remove the column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur Perform the optional centrifugatio
12. on Handling RNA 42 Appendix B Storage Quantification and Determination of Quality of RNA 44 Appendix C Protocol for Formaldehyde Agarose Gel Electrophoresis 47 Appendix D Storage Quantification and Determination of Quality of Genomic DNA 49 Appendix E Optional On Column DNase Digestion Using the RNase Free DNase Set 51 Appendix F Quantification of Protein in SDS PAGE Sample Buffer 53 Ordering Information 54 AllPrep DNA RNA Protein Mini Handbook 09 2011 3 Kit Contents AllPrep DNA RNA Protein Mini Kit 50 Catalog no 80004 Number of preps 50 AllPrep DNA Mini Spin Columns uncolored 50 each in a 2 ml Collection Tube RNeasy Mini Spin Columns pink 50 each in a 2 ml Collection Tube Collection Tubes 1 5 ml 100 Collection Tubes 2 ml 150 Buffer RLT 45 ml Buffer RW1 45 ml Buffer RPE concentrate 11ml RNase Free Water 10 ml Buffer AW1 t concentrate 19 ml Buffer AW2 concentrate 13 ml Buffer EB 22 ml Buffer APP 55 ml Buffer ALO 10 ml Handbook 1 Contains a guanidine salt Not compatible with disinfectants containing bleach See page 6 for safety information Before using for the first time add the appropriate volume of ethanol 96 100 as indicated on the bottle to obtain a working solution DTT must be added before use See Things to do before starting in the protocols Storage The AllPrep DNA RNA Protein Mini Kit should be stored dry at room temperature 15 25 C and is
13. stabilization and for tissue disruption and homogenization in the Allprotect Tissue Reagent Handbook before proceeding to step 4 of this protocol Otherwise start at step 2 below 2 Excise the tissue sample from the animal or remove it from storage Determine the amount of tissue Do not use more than 30 mg Proceed immediately to step 3 Weighing tissue is the most accurate way to determine the amount If necessary cut the tissue on a clean surface and weigh the piece to be used RNA and proteins in harvested tissues are not protected until the tissues are treated with Allprotect Tissue Reagent flash frozen or disrupted and homogenized in step 3 Frozen tissues should not be allowed to thaw during handling The relevant procedures should be carried out as quickly as possible Note Remaining fresh tissues can be placed into Allprotect Tissue Reagent to stabilize DNA RNA and proteins see the Allprotect Tissue Handbook However previously frozen tissues thaw too slowly in the reagent preventing the reagent from diffusing into the tissues quickly enough to prevent RNA and protein degradation 3 Disrupt the tissue and homogenize the lysate in Buffer RLT do not use more than 30 mg tissue according to step 3a 3b 3c or 3d See Disrupting and homogenizing starting material page 14 for more details on disruption and homogenization Note Ensure that B ME is added to Buffer RLT before use see Things to do before starting
14. to wash the spin column membrane Discard the flow through Reuse the collection tube in step 9 Note After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Be sure to empty the collection tube completely Optional If purifying RNA from tissues with high DNA content and if the RNA will be used in sensitive downstream applications we recommend performing DNase digestion by following steps E1 E4 Appendix E page 51 instead of step 8 Add 500 pl Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 15 s at gt 8000 x g 210 000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in step 10 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting Add 500 pl Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 2 min at gt 8000 x g 210 000 rpm to wash the spin column membrane The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during RNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur Flow through contains Buffer RLT or Bu
15. you are familiar with operating it by referring to the TissueRuptor User Manual and TissueRuptor Handbook RNAlater RNA Stabilization Reagent which stabilizes RNA only can be used instead of Allprotect Tissue Reagent if protein will not be purified AllPrep DNA RNA Protein Mini Handbook 09 2011 27 If using the TissueLyser ensure that you are familiar with operating it by referring to the operating instructions and Tissuelyser Handbook For optimal results stabilize harvested tissues immediately in Allprotect Tissue Reagent see the Allprotect Tissue Reagent Handbook Tissues can be stored in the reagent for up to 1 day at 37 C 7 days at 15 25 C or 6 months at 2 8 C or archived at 20 C or 80 C Fresh frozen or Allprotect stabilized tissues can be used Tissues can be stored at 70 C for several months Flashfreeze tissues in liquid nitrogen and immediately transfer to 70 C Do not allow tissues to thaw during weighing or handling prior to disruption in Buffer RLT Homogenized tissue lysates from step 3 can also be stored at 70 C for several months Incubate frozen lysates at 37 C in a water bath until completely thawed and salts are dissolved before continuing with step 4 Avoid prolonged incubation which may compromise RNA integrity If desired more than 30 mg tissue can be disrupted and homogenized at the start of the procedure increase the volume of Buffer RLT proportionately Use a portion of the homog
16. 04 Collection Tubes RNase Free Reagents and Buffers RNeasy Fibrous Tissue Mini Kit for purification of up to 100 pg total RNA from fiber rich tissues RNeasy Fibrous Tissue 50 RNeasy Mini Spin Columns 74704 Mini Kit 50 Collection Tubes Proteinase K RNase Free DNase RNase Free Reagents and Buffers Related products for total protein preparation Qproteome Mammalian Protein Prep Kit for total protein preparations from mammalian cells and tissues Qproteome Mammalian For approximately 100 protein 37901 Protein Prep Kit preparations from cultured mammalian cells Buffer Reagents Protease Inhibitor Solution Benzonase Larger kit sizes and or formats available see www giagen com 56 AllPrep DNA RNA Protein Mini Handbook 09 2011 Ordering Information Product Contents Cat no Related products for PCR and RT PCR applications QIAGEN Fast Cycling PCR Kit for fast and specific PCR on any thermal cycler QIAGEN Fast Cycling For 200 x 20 pl reactions 2x 1 ml 203743 PCR Kit 200 QIAGEN Fast Cycling PCR Master Mix 10x Coralload Fast Cycling Dye Q Solution RNase Free Water QIAGEN Multiplex PCR Kit for highly specific and sensitive multiplex PCR without optimization QIAGEN Multiplex PCR For 100 x 50 pl reactions 1 7 ml 206143 Kit 100 2x Master Mix 5x Q Solution 2 x 1 7 ml RNase Free Water QIAGEN OneStep RT PCR Kit for fast and successful one step RT PCR QIAGEN OneStep RT PCR For 25 x 50 pl reacti
17. 2 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN goes Sample amp Assay Technologies
18. 5 20 10 20 800 Amounts can vary depending on the disruption and homogenization method see page 14 Amounts can vary due to factors such as species developmental stage and growth conditions Since the AllPrep DNA RNA Protein procedure enriches for mRNA and other RNA species gt 200 nucleotides the total RNA yield does not include 5S rRNA tRNA and other low molecular weight RNAs which make up 15 20 of total cellular RNA AllPrep DNA RNA Protein Mini Handbook 09 2011 13 Handling and storing starting material RNA and protein in harvested tissue are not protected until the sample is treated with Allprotect Tissue Reagent flashfrozen or disrupted and homogenized in the presence of RNase inhibiting or denaturing reagents Otherwise unwanted changes in the gene expression profile at both the mRNA and protein levels will occur It is therefore important that tissue samples are immediately frozen in liquid nitrogen and stored at 70 C or immediately immersed in Allprotect Tissue Reagent The procedures for tissue harvesting and RNA and protein protection should be carried out as quickly as possible Frozen tissue samples should not be allowed to thaw during handling or weighing After disruption and homogenization in Buffer RLT lysis buffer samples can be stored at 70 C for months Disrupting and homogenizing starting material Efficient disruption and homogenization of the starting material is an absolute requirement
19. 500 pl Buffer AW1 to the AllPrep DNA spin column from step 5 Close the lid gently and centrifuge for 15 s at gt 8000 x g 10 000 rpm to wash the spin column membrane Discard the flow through Reuse the spin column in step 22 Note Buffer AW1 is supplied as a concentrate Ensure that ethanol is added to Buffer AW 1 before use see Things to do before starting Flow through contains Buffer AW1 and is therefore not compatible with bleach See page 6 for safety information AllPrep DNA RNA Protein Mini Handbook 09 2011 25 22 23 24 26 Add 500 pl Buffer AW2 to the AllPrep DNA spin column Close the lid gently and centrifuge for 2 min at full speed to wash the spin column membrane Note Buffer AW2 is supplied as a concentrate Ensure that ethanol is added to Buffer AW2 before use see Things to do before starting The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during DNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the AllPrep DNA spin column from the collection tube If the column contacts the flow through empty the collection tube and centrifuge the spin column again for 1 min at full speed Place the AllPrep DNA spin column in a new 1 5 ml collection tube supplied Add 100 pl Buffer EB preheated to 70 C directly to the spin column membrane and close the lid Incubate at room t
20. 72 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 080 000 7146 Fax 02 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1 122 330 Technical 01 800 7742 436 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7050 Fax 911 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 92
21. A spin column Close the lid gently and centrifuge for 2 min at full speed to wash the spin column membrane Note Buffer AW2 is supplied as a concentrate Ensure that ethanol is added to Buffer AW2 before use see Things to do before starting Flow through contains Buffer AW1 and is therefore not compatible with bleach See page 6 for safety information 34 AllPrep DNA RNA Protein Mini Handbook 09 2011 The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during DNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the AllPrep DNA spin column from the collection tube If the column contacts the flow through empty the collection tube and centrifuge the spin column again for 1 min at full speed 23 Place the AllPrep DNA spin column in a new 1 5 ml collection tube supplied Add 100 pl Buffer EB preheated to 70 C directly to the spin column membrane and close the lid Incubate at room temperature 15 25 C for 2 min and then centrifuge for 1 min at gt 8000 x g 10 000 rpm to elute the DNA 24 Repeat step 23 to elute further DNA To prevent dilution of the first DNA eluate use a new 1 5 ml collection tube not supplied to collect the second DNA eluate To combine the first and second DNA eluates reuse the collection tube from step 23 Note To achieve a higher DNA concentration elute with 2 x 50 pl Buffer EB The
22. Irritating to eyes and skin R52 53 Harmful to aquatic organisms and may cause long term adverse effects to the aquatic environment S13 Keep away from food drink and animal feedingstuffs S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 Wear suitable protective clothing S46 If swallowed seek medical advice immediately and show the container or label 6 AllPrep DNA RNA Protein Mini Handbook 09 2011 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of AllPrep DNA RNA Protein Mini Kit is tested against predetermined specifications to ensure consistent product quality AllPrep DNA RNA Protein Mini Handbook 09 2011 7 Introduction The AllPrep DNA RNA Protein Mini Kit is designed to purify genomic DNA total RNA and total protein simultaneously from a single biological sample In contrast to other procedures the kit allows maximal recovery of DNA RNA and protein There is no need to divide the sample into 3 before purifying DNA RNA and protein separately There is also no need to purify total nucleic acids first and then to divide the purified nucleic acids into 2 before purifying DNA and RNA separately The kit is compatible with small am
23. September 2011 AllPrep DNA RNA Protein Mini Handbook For simultaneous purification of genomic DNA total RNA and total protein from the same cell or tissue sample QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Storage 4 Product Use Limitations 5 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Safety Information 6 Quality Control 7 Introduction 8 Principle and procedure 9 Equipment and Reagents to Be Supplied by User 11 Important Notes 12 Determining the amount of starting material 12 Handling and storing starting material 14 Disrupting and homogenizing starting material 14 Protocols H Simultaneous Purification of Genomic DNA Total RNA and Total Protein from Animal and Human Cells 18 E Simultaneous Purification of Genomic DNA Total RNA and Total Protein from Animal and Human Tissues 27 Troubleshooting Guide 36 Appendix A General Remarks
24. al centrifugation to dry the RNeasy spin column membrane if any flow through is present on the outside of the column step 11 of the protocols When processing cultured cells ensure complete removal of cell culture medium after harvesting cells see protocol page 18 Add ethanol to the lysate after passing the lysate through the AllPrep DNA spin column The final homogenate should have a pH of 7 Make sure that the sample is not highly acidic or basic Contamination of RNA with DNA affects downstream applications a Cell number too high For some cell types the efficiency of DNA binding to the AllPrep DNA spin column may be reduced when processing very high cell numbers If the eluted RNA contains substantial DNA contamination try processing smaller cell numbers AllPrep DNA RNA Protein Mini Handbook 09 2011 37 Comments and suggestions b Incomplete removal of cell culture medium or stabilization reagent c Tissue has high DNA content Low Az 0 Azs value in RNA eluate Water used to dilute RNA for A260 A280 measurement RNA degraded a Inappropriate handling of starting material Be sure to remove any excess cell culture medium or stabilization reagent to prevent significant dilution of the lysis buffer The AllPrep DNA spin column will not bind DNA effectively if the lysis buffer is diluted For certain tissues with extremely high DNA content e g thymus some DNA will pass through the AllPre
25. and proteins see page 14 10 AllPrep DNA RNA Protein Mini Handbook 09 2011 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier For all protocols 14 3 M B mercaptoethanol ME commercially available solutions are usually 14 3 M Dithiothreitol DTT Sterile RNase free pipet tips Microcentrifuge with rotor for 2 ml tubes 96 100 ethanol 70 ethanol in water Disposable gloves For tissue samples Allprotect Tissue Reagent see ordering information page 54 or liquid nitrogen Equipment for sample disruption and homogenization see pages 14 17 Depending on the method chosen one or more of the following are required Trypsin and PBS QlAshredder homogenizer see ordering information page 54 Blunt ended needle and syringe Mortar and pestle TissueLyser see ordering information page 54 TissueRuptor see ordering information page 54 Optional 5 w v sodium dodecyl sulfate SDS or 8 M urea for details see the protocols Do not use denatured alcohol which contains other substances such as methanol or methylethylketone AllPrep DNA RNA Protein Mini Handbook 09 2011 11 Important Notes Determining the amount of starting material It is essential fo use the correct amount of s
26. ate 10 mM EDTA pH to 7 0 with NaOH 1x FA gel running buffer 100 ml 10x FA gel buffer 20 ml 37 12 3 M formaldehyde 880 ml RNase free water 5x RNA loading buffer 16 pl saturated aqueous bromophenol blue solution 80 pl 500 mM EDTA pH 8 0 720 pl 37 12 3 M formaldehyde 2 ml 100 glycerol 3 084 ml formamide 4 ml 10x FA gel buffer RNase free water to 10 ml Stability approximately 3 months at 4 C When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier To make a saturated solution add solid bromophenol blue to distilled water Mix and continue to add more bromophenol blue until no more will dissolve Centrifuge to pellet the undissolved powder and carefully pipet the saturated supernatant 48 AllPrep DNA RNA Protein Mini Handbook 09 2011 Appendix D Storage Quantification and Determination of Quality of Genomic DNA Storage of DNA For long term storage purified DNA in Buffer EB can be stored at 20 C Avoid any contamination as this may lead to DNA degradation We recommend storing samples in aliquots in order to avoid repeated freezing and thawing which can cause formation of precipitates Quantification of DNA DNA concentration can be determined by measuring the absorbance at 260 nm Aso in a spectrophotometer using a quartz cuvette
27. column membrane are performed according to the protocol starting on page 27 After washing with a reduced volume of Buffer RW1 RNA is treated with DNase while bound to the spin column membrane DNase is removed by a second wash with Buffer RW1 Washing with Buffer RPE and elution are then performed according to the protocol on page 27 Important points before starting E Do not vortex the reconstituted DNase I DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the vial Things to do before starting E Prepare DNase stock solution before using the RNase Free DNase Set for the first time Dissolve the solid DNase 1500 Kunitz units in 550 pl of the RNase free water provided To avoid loss of DNase do not open the vial Inject RNase free water into the vial using an RNase free needle and syringe Mix gently by inverting the vial Do not vortex E For long term storage of DNase l remove the stock solution from the glass vial divide it into single use aliquots and store at 20 C for up to 9 months Thawed aliquots can be stored at 2 8 C for up to 6 weeks Do not refreeze the aliquots after thawing AllPrep DNA RNA Protein Mini Handbook 09 2011 51 Procedure Carry out the protocol starting on page 27 up to and including step 7 Instead of peforming step 8 the wash with Buffer RW 1 follow steps E1 E4 below El E2 E3 E4 Add 350 pl Buffer RW1 to t
28. d productspecific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor Larger kit sizes also available see www giagen com For real time PCR two step RT PCR and one step RT PCR with sequence specific probes QuantiFast Probe Kits are available see www giagen com FastPCR 58 AllPrep DNA RNA Protein Mini Handbook 09 2011 Trademarks QIAGEN AllPrep DNeasy MinElute Qproteome QuantiFast Quantiscript QuantiTect RNeasy TissueRuptor QIAGEN Group Agilent Agilent Technologies Inc Benzonase Merck KGaA Germany Applied Biosystems SYBR Life Technologies Corporation LightCycler Roche Group RNAlater is a trade mark of AMBION Inc Austin Texas and is covered by various U S and foreign patents Benzonase Nuclease is supplied by Merck KGaA and its Affiliates Benzonase is a registered trademark of Merck KGaA Darmstadt Germany Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the AllPrep DNA RNA Protein Mini Kit to the following terms 1 The AllPrep DNA RNA Protein Mini Kit may be used solely in accordance with the AllPrep DNA RNA Protein Mini Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual pr
29. des fast cDNA synthesis with integrated removal of genomic DNA contamination see ordering information page 57 Integrity of RNA The integrity and size distribution of total RNA purified with the AllPrep DNA RNA Protein Mini Kit can be checked by denaturing agarose gel electrophoresis and ethidium bromide staining or by using an Agilent 2100 bioanalyzer The respective ribosomal RNAs should appear as sharp bands or peaks The apparent ratio of 28S rRNA to 18S rRNA should be approximately 2 1 If the ribosomal bands or peaks of a specific sample are not sharp but appear as a smear towards smaller sized RNAs it is likely that the sample suffered major degradation either before or during RNA purification When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 46 AllPrep DNA RNA Protein Mini Handbook 09 2011 Appendix C Protocol for Formaldehyde Agarose Gel Electrophoresis The following protocol for formaldehyde agarose FA gel electrophoresis is routinely used at QIAGEN and gives enhanced sensitivity for gel and subsequent analysis e g northern blotting A key feature is the concentrated RNA loading buffer that allows a larger volume of RNA sample to be loaded onto the gel than conventional protocols e g Sambrook et al eds 1989 Molecular cloning a labo
30. dissolved Avoid prolonged incubation which may compromise RNA integrity If any insoluble material is visible centrifuge for 5 min at 3000 5000 x g Transfer supernatant to a new RNase free glass or polypropylene tube and continue with step 4 AllPrep DNA RNA Protein Mini Handbook 09 2011 19 Buffer RLT Buffer RW1 and Buffer AW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 6 for safety information Perform all steps of the procedure at room temperature 15 25 C During the procedure work quickly Perform all centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C Things to do before starting HM 6 Mercaptoethanol B ME must be added to Buffer RLT before use Add 10 pl B ME per 1 ml Buffer RLT Dispense in a fume hood and wear appropriate protective clothing Buffer RLT containing B ME can be stored at room temperature 15 25 C for up to 1 month Dithiothreitol DTT must be added to Buffer ALO before use Add 8 mg DTT per 1 ml Buffer ALO M Buffer RPE Buffer AW1 and Buffer AW2 are each supplied as a concentrate Before using for the first time add the appropriate volume of ethanol 96 100 as indicated on the bottle to obtain a working solution Buffer RLT may form a precipitate during storage If necessary redissolve by warming and then place at room temperature M Preheat Buff
31. e homogenate stand at room temperature for 2 3 min until the foam subsides before continuing with the procedure 3b Disruption and homogenization using the TissueLyser Place the tissues in 2 ml microcentrifuge tubes containing 1 stainless steel bead 5 mm mean diameter If handling fresh or frozen tissue samples keep the tubes on dry ice Place the tubes at room temperature Immediately add the appropriate volume of Buffer RLT see Table 7 per tube Place the tubes in the TissueLyser Adapter Set 2 x 24 Operate the TissueLyser for 2 min at 20 Hz The time depends on the tissue being processed and can be extended until the tissue is completely homogenized Rearrange the collection tubes so that the outermost tubes are innermost and the innermost tubes are outermost Operate the TissueLyser for another 2 min at 20 Hz Rearranging the tubes allows even homogenization Proceed to step 4 Do not reuse the stainless steel beads 3c Disruption using a mortar and pestle followed by homogenization using a QlAshredder homogenizer m Immediately place the weighed tissue in liquid nitrogen and grind thoroughly with a mortar and pestle m Decant tissue powder and liquid nitrogen into an RNase free liquid nitrogen cooled 2 ml microcentrifuge tube not supplied Allow the liquid nitrogen to evaporate but do not allow the tissue to thaw 30 AllPrep DNA RNA Protein Mini Handbook 09 2011 m Add the appropriate volume of Buffer
32. elds will not be consistent and less than expected If lysis of the starting material is incomplete DNA and RNA yields will be lower than expected even if the binding capacity of the spin columns is not exceeded 12 AllPrep DNA RNA Protein Mini Handbook 09 2011 Table 1 Specifications of the spin columns in the AllPrep DNA RNA Protein Mini Kit AllPrep DNA spin RNeasy spin column Specification column Maximum binding capacity 100 pg DNA 100 pg RNA Maximum loading volume 700 pl 700 pl Nucleic acid size distribution DNA of 15 30 kbt RNA gt 200 nucleotides Minimum elution volume 100 pl 30 pl Maximum amount of starting material E Animal and human cells 1 x 10 cells Entire flow through from AllPrep DNA spin column E Animal and human tissues 30 mg Entire flow through from AllPrep DNA spin column Loading more than 20 pg DNA may lead to DNA contamination of the RNA eluate t Depending on homogenization conditions If using Allprotect stabilized tissues 15 20 mg tissue should be used Table 2 Typical yields of genomic DNA total RNA and total protein with the AllPrep DNA RNA Protein Mini Kit Typical yield pg of Sample type Genomic DNA Total RNA Total protein Cell cultures 1 x 10 cells E NIH 3T3 8 10 50 Hela Jurkat 6 15 60 E COS 7 7 35 150 Mouse rat tissues 10 mg E Kidney 15 25 20 30 1300 E Liver 15 25 40 60 1000 E Spleen 50 70 30 80 1000 E Thymus 50 100 40 80 800 E Lung 1
33. emperature 15 25 C for 2 min and then centrifuge for 1 min at gt 8000 x g 10 000 rpm to elute the DNA Repeat step 23 to elute further DNA To prevent dilution of the first DNA eluate use a new 1 5 ml collection tube not supplied to collect the second DNA eluate To combine the first and second DNA eluates reuse the collection tube from step 23 Note To achieve a higher DNA concentration elute with 2 x 50 pl Buffer EB The final DNA yield however may be reduced AllPrep DNA RNA Protein Mini Handbook 09 2011 Protocol Simultaneous Purification of Genomic DNA Total RNA and Total Protein from Animal and Human Tissues Determining the correct amount of starting material Itis essential to use the correct amount of starting material in order to obtain optimal nucleic acid yield and purity A maximum amount of 30 mg fresh or frozen tissue or 15 20 mg Allprotect stabilized tissue can generally be processed For most tissues the DNA binding capacity of the AllPrep DNA spin column the RNA binding capacity of the RNeasy spin column and the lysing capacity of Buffer RLT will not be exceeded by these amounts However smaller amounts may allow more efficient separation of DNA and RNA Average DNA and RNA yields from various tissues are given in Table 2 page 13 Some tissues such as spleen and thymus contain very high amounts of DNA which will overload the AllPrep DNA spin column unless less than 5 mg tissue is used as startin
34. enate corresponding to no more than 30 mg tissue for nucleic acid purification and store the rest at 80 C Buffer RLT Buffer RW1 and Buffer AW1 contain a guanidine salt and are therefore not compatible with disinfecting reagents containing bleach See page 6 for safety information Perform all steps of the procedure at room temperature During the procedure work quickly Perform all centrifugation steps at 20 25 C in a standard microcentrifuge Ensure that the centrifuge does not cool below 20 C Things to do before starting 28 B Mercaptoethanol B ME must be added to Buffer RLT before use Add 10 pl B ME per 1 ml Buffer RLT Dispense in a fume hood and wear appropriate protective clothing Buffer RLT containing B ME can be stored at room temperature 15 25 C for up to 1 month Dithiothreitol DTT must be added to Buffer ALO before use Add 8 mg DTT per 1 ml Buffer ALO Buffer RPE Buffer AW1 and Buffer AW2 are each supplied as a concentrate Before using for the first time add the appropriate volume of ethanol 96 100 as indicated on the bottle to obtain a working solution Buffer RLT may form a precipitate upon storage If necessary redissolve by warming and then place at room temperature Preheat Buffer EB to 70 C to ensure optimal DNA elution AllPrep DNA RNA Protein Mini Handbook 09 2011 Procedure Sample disruption and homogenization 1 If using Allprotect Tissue Reagent follow the protocols for tissue
35. end using either QIAGEN Genomic ips or Blood amp Cell Culture DNA Kits Both allow purification of DNA of up to 150 kb in size See page 55 for ordering information Visit www giagen com geneXpression for information on standardized solutions for gene expression analysis from QIAGEN 8 AllPrep DNA RNA Protein Mini Handbook 09 2011 E Poly A RNA selection HM RNase S1 nuclease protection E Microarrays Proteins recovered with the AllPrep DNA RNA Protein procedure are suitable for downstream applications such as M 1D and 2D gel electrophoresis E Western blotting Principle and procedure The AllPrep DNA RNA Protein procedure integrates QIAGEN s patented technology for selective binding of double stranded DNA with well established RNeasy technology and combines this with a new protein precipitation chemistry Efficient purification of high quality DNA RNA and proteins is guaranteed without the need for additional RNase and DNase digestions Biological samples are first lysed and homogenized in a highly denaturing guanidine isothiocyanate containing buffer which immediately inactivates DNases and RNases as well as proteases to ensure isolation of intact DNA RNA and proteins The lysate is then passed through an AllPrep DNA spin column This column in combination with the high salt buffer allows selective and efficient binding of genomic DNA The column is washed and pure ready to use DNA is then eluted Etha
36. er EB to 70 C to ensure optimal DNA elution Procedure Sample disruption and homogenization 1 la 20 Harvest cells according to step 1a or 1b Cells grown in suspension do not use more than 1 x 10 cells Determine the number of cells Pellet the appropriate number of cells by centrifuging for 5 min at 300 x g in a centrifuge tube not supplied Carefully remove all supernatant by aspiration and proceed to step 2 Note Incomplete removal of cell culture medium will inhibit lysis and dilute the lysate affecting the conditions for nucleic acid purification Both effects may reduce nucleic acid yields and purity Cells grown in a monolayer do not use more than 1 x 10 cells Cells grown in a monolayer in cell culture vessels can be either lysed directly in the vessel up to 10 cm diameter or trypsinized and collected as a cell pellet prior to lysis Cells grown in a monolayer in cell culture flasks should always be trypsinized AllPrep DNA RNA Protein Mini Handbook 09 2011 To lyse cells directly Determine the number of cells Completely aspirate the cell culture medium and proceed immediately to step 2 Note Incomplete removal of cell culture medium will inhibit lysis and dilute the lysate affecting the conditions for nucleic acid purification Both effects may reduce nucleic acid yields and purity To trypsinize and collect cells Determine the number of cells Aspirate the medium and wash the cells w
37. ffer RW1 and is therefore not compatible with bleach See page 6 for safety information 32 AllPrep DNA RNA Protein Mini Handbook 09 2011 Optional Place the RNeasy spin column in a new 2 ml collection tube supplied and discard the old collection tube with the flow through Centrifuge at full speed for 1 min Perform this step to eliminate any possible carryover of Buffer RPE or if residual flow through remains on the outside of the RNeasy spin column after step 10 Place the RNeasy spin column in a new 1 5 ml collection tube supplied Add 30 50 pl RNase free water directly to the spin column membrane Close the lid gently and centrifuge for 1 min at gt 8000 x g gt 10 000 rpm to elute the RNA If the expected RNA yield is gt 30 pg repeat step 12 using another 30 50 pl of RNase free water or using the eluate from step 12 if high RNA concentration is required Reuse the collection tube from step 12 If using the eluate from step 12 the RNA yield will be 15 30 less than that obtained using a second volume of RNase free water but the final RNA concentration will be higher Total protein precipitation 14 15 16 Add 1 volume usually 600 pl or 1000 pl of Buffer APP to the flow through from step 7 Mix vigorously and incubate at room temperature for 10 min to precipitate protein Centrifuge at full speed for 10 min and carefully decant the supernatant Add 500 pl of 70 ethanol to the protein pe
38. for all nucleic acid purification procedures Disruption and homogenization are 2 distinct steps MH Disruption Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the nucleic acids contained in the sample Different samples require different methods to achieve complete disruption Incomplete disruption results in significantly reduced nucleic acid yields M Homogenization Homogenization is necessary to reduce the viscosity of the lysates produced by disruption Homogenization shears high molecular weight cellular components to create a homogeneous lysate Incomplete homogenization results in inefficient binding of DNA and RNA and therefore significantly reduced yield and purity of nucleic acids Excessive homogenization on the other hand results in shorter genomic DNA fragments Some mechanical homongenization methods other than the TissueRuptor and Tissuelyser may lead to protein degradation due to prolonged warming of the homogenate Some disruption methods simultaneously homogenize the sample while others require an additional homogenization step Table 3 gives an overview of various disruption and homogenization methods and is followed by a detailed description of each method RNAlater RNA Stabilization Reagent which stabilizes RNA only can be used instead if proteins will not be purified 14 AllPrep DNA RNA Protein Mini Handbook 09 2011 Table 3 Disruption and homogenizat
39. g material For these tissues we recommend performing DNase digestion on the RNeasy spin column membrane if the eluted RNA will be used in downstream applications sensitive to very small amounts of DNA for further details see Appendix E page 51 RNA yields from skeletal muscle heart and skin tissue may be low due to the abundance of contractile proteins connective tissue and collagen For purification of genomic DNA and total RNA from these tissues we recommend using the DNeasy Blood amp Tissue Kit and the RNeasy Fibrous Tissue Mini Kit respectively see pages 55 and 56 for ordering information If there is no information about the nature of your starting material we recommend starting with no more than 10 mg tissue Depending on nucleic acid yield and purity it may be possible to use up to 30 mg tissue in subsequent preparations Do not overload the AllPrep DNA spin column as this will lead to copurification of DNA with RNA Do not overload the RNeasy spin column as this will significantly reduce RNA yield and purity Weighing tissue is the most accurate way to quantitate the amount of starting material As a guide a 3 mm cube 27 mm of most animal tissues weighs 30 35 mg Important points before starting HM If using the AllPrep DNA RNA Protein Mini Kit for the first time read Important Notes page 12 E f preparing RNA for the first time read Appendix A page 42 E f using the TissueRuptor ensure that
40. g pretreatment and use of disposable and nondisposable vessels and solutions while working with RNA General handling Proper microbiological aseptic technique should always be used when working with RNA Hands and dust particles may carry bacteria and molds and are the most common sources of RNase contamination Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin or from dusty laboratory equipment Change gloves frequently and keep tubes closed whenever possible Keep purified RNA on ice when aliquots are pipetted for downstream applications Disposable plasticware The use of sterile disposable polypropylene tubes is recommended throughout the procedure These tubes are generally RNase free and do not require pretreatment to inactivate RNases Nondisposable plasticware Nondisposable plasticware should be treated before use to ensure that it is RNase free Plasticware should be thoroughly rinsed with 0 1 M NaOH 1 mM EDTA followed by RNase free water see Solutions page 43 Alternatively chloroform resistant plasticware can be rinsed with chloroform to inactivate RNases When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 42 AllPrep DNA RNA Protein Mini Handbook 09 2011
41. he RNeasy spin column and centrifuge for 15 s at 28000 x g 210 000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in step E4 Add 10 pl DNase I stock solution see above to 70 pl Buffer RDD Mix by gently inverting the tube and centrifuge briefly to collect residual liquid from the sides of the tube Buffer RDD is supplied with the RNase Free DNase Set Note DNase is especially sensitive to physical denaturation Mixing should only be carried out by gently inverting the tube Do not vortex Add the DNase incubation mix 80 pl directly to the RNeasy spin column membrane and place on the benchtop 20 30 C for 15 min Note Be sure to add the DNase incubation mix directly to the RNeasy spin column membrane DNase digestion will be incomplete if part of the mix sticks to the walls or the O ring of the spin column Add 350 pl Buffer RW1 to the RNeasy spin column and centrifuge for 15 s at 28000 x g 210 000 rpm Discard the flow through Continue with step 9 of the protocol on page 27 i e the first wash with Buffer RPE Reuse the collection tube in step 9 Flow through contains Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information 52 AllPrep DNA RNA Protein Mini Handbook 09 2011 Appendix F Quantification of Protein in SDS PAGE Sample Buffer The strong denaturation conditions in the AllPrep DNA RNA Protein procedure mean that
42. ion dries the spin column membrane ensuring that no ethanol is carried over during RNA elution Residual ethanol may interfere with downstream reactions Flow through contains Buffer RLT or Buffer RW1 and is therefore not compatible with bleach See page 6 for safety information AllPrep DNA RNA Protein Mini Handbook 09 2011 23 Note After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur Optional Place the RNeasy spin column in a new 2 ml collection tube supplied and discard the old collection tube with the flow through Centrifuge at full speed for 1 min Perform this step to eliminate any possible carryover of Buffer RPE or if residual flow through remains on the outside of the RNeasy spin column after step 10 Place the RNeasy spin column in a new 1 5 ml collection tube supplied Add 30 50 pl RNase free water directly to the spin column membrane Close the lid gently and centrifuge for 1 min at gt 8000 x g 210 000 rpm to elute the RNA If the expected RNA yield is gt 30 pg repeat step 12 using another 30 50 pl of RNase free water or using the eluate from step 12 if high RNA concentration is required Reuse the collection tube from step 12 If using the eluate from step 12 the RNA yield will be 15 30 less than that obtained using a second volume of RNase free water but
43. ion methods Sample Disruption method Homogenization method Cells Addition of lysis buffer TissueRuptor or QlAshredder homogenizer or syringe and needle Tissues TissueRuptor TissueRuptor TissueLyser TissueLyser Mortar and pestle QlAshredder homogenizer or syringe and needle Simultaneously disrupts and homogenizes individual samples t Simultaneously disrupts and homogenizes up to 192 samples in parallel Results are comparable to those obtained using the TissueRuptor or other rotor stator homogenizer Disruption and homogenization using the TissueRuptor The TissueRuptor is a rotor stator homogenizer that thoroughly disrupts and simultaneously homogenizes single tissue samples in the presence of lysis buffer in 15 90 seconds depending on the toughness and size of the sample The TissueRuptor can also be used to homogenize cell lysates The blade of the TissueRuptor disposable probe rotates at a very high speed causing the sample to be disrupted and homogenized by a combination of turbulence and mechanical shearing For guidelines on using the TissueRuptor refer to the TissueRuptor Handbook For other rotor stator homogenizers refer to suppliers guidelines Note Longer homogenization times with the TissueRuptor result in greater DNA fragmentation or even protein degradation due to increasing temperature Therefore the homogenization time should be kept as short as possible if the DNA will be used in downs
44. ith PBS Aspirate the PBS and add 0 10 0 25 trypsin in PBS After the cells detach from the dish or flask add medium containing serum to inactivate the trypsin transfer the cells to an RNase free glass or polypropylene centrifuge tube not supplied and centrifuge at 300 x g for 5 min Completely aspirate the supernatant and proceed to step 2 Note Incomplete removal of cell culture medium will inhibit lysis and dilute the lysate affecting the conditions for nucleic acid purification Both effects may reduce nucleic acid yields and purity 2 Disrupt the cells by adding Buffer RLT For pelleted cells loosen the cell pellet thoroughly by flicking the tube Add the appropriate volume of Buffer RLT see Table 5 Vortex or pipet to mix and proceed to step 3 Note Incomplete loosening of the cell pellet may lead to inefficient lysis and reduced nucleic acid yields Ensure that B ME is added to Buffer RLT before use see Things to do before starting Table 5 Volumes of Buffer RLT for lysing pelleted cells Number of pelleted cells Volume of Buffer RLT lt 5 x 10 350 pl 5x 10 1x 107 600 pl For direct lysis of cells grown in a monolayer add the appropriate volume of Buffer RLT see Table 6 to the cell culture dish Collect the lysate with a rubber policeman Pipet the lysate into a microcentrifuge tube not supplied Vortex or pipet to mix and ensure that no cell clumps are visible before proceeding to ste
45. llet Centrifuge at full speed for 1 min and remove the supernatant by using a pipet or by decanting as much liquid as possible It is not necessary to resupend or incubate the pellet Dry the protein pellet for 5 10 min at room temperature Note Incomplete drying may cause problems when loading the protein onto a gel due to residual ethanol Add up to 100 pl Buffer ALO and mix vigorously to dissolve the protein pellet Note Ensure that DTT is added to Buffer ALO before use see Things to do before starting The volume of Buffer ALO to add depends on the amount of starting material See Table 2 page 13 for typical protein yields from various starting materials Buffer ALO is a Laemmli related sample buffer for use in SDS PAGE and contains bromophenol blue dye If the proteins will not be analyzed by SDS PAGE dissolve the pellet in a buffer compatible with the intended downstream application Supernatant contains Buffer RLT and is therefore not compatible with bleach See page 6 for safety information AllPrep DNA RNA Protein Mini Handbook 09 2011 33 Due to the strong denaturing conditions with Buffer RLT which is necessary to inactivate RNases and proteases the precipitated proteins may show reduced solubility Vortex for several minutes or disaggregate the pellet by pipetting up and down several times Depending on the sample type the pellet may contain proteins or other cellular components that are not so
46. ls RNA content can vary greatly between cell types The following examples illustrate how to determine the maximum amount of starting material HM COS cells have high RNA content approximately 35 pg RNA per 10 cells Do not use more than 3 x 10 cells otherwise the RNA binding capacity of the RNeasy spin column will be exceeded M Hela cells have average RNA content approximately 15 pg RNA per 10 cells Do not use more than 7 x 10 cells otherwise the RNA binding capacity of the RNeasy spin column will be exceeded E NIH 3T3 cells have low RNA content approximately 10 pg RNA per 10 cells The maximum amount of starting material 1 x 107 cells can be used If processing a cell type not listed in Table 2 page 13 and if there is no information about its RNA content we recommend starting with no more than 3 4 x 104 cells Depending on RNA yield and purity it may be possible to increase the cell number in subsequent preparations Do not overload the AllPrep DNA spin column as this will lead to copurification of DNA with RNA Do not overload the RNeasy spin column as this will significantly reduce RNA yield and purity Counting cells is the most accurate way to quantitate the amount of starting material As a guide the number of Hela cells obtained in various culture vessels after confluent growth is given in Table 4 18 AllPrep DNA RNA Protein Mini Handbook 09 2011 Table 4 Growth area and number of Hela cells in va
47. luble For details on how to solubilize these samples see Troubleshooting Guide on page 36 For easier dissolving or if the proteins need to be quantified prior to SDS PAGE dissolve the pellet in 5 w v SDS or 8 M urea For details about protein quantification see Appendix F page 53 Buffer ALO may turn yellow upon dissolving protein but this has no effect on downstream applications The buffer will turn blue again in SDS PAGE sample buffer If using a stock solution of your own sample buffer e g 5x concentration be sure to dilute it accordingly so that the protein sample to be loaded on a gel has a 1x concentration of sample buffer 19 Incubate for 5 min at 95 C to completely dissolve and denature protein Then cool the sample to room temperature 20 Centrifuge for 1 min at full speed to pellet any residual insoluble material Use the supernatant in downstream applications such as SDS PAGE and western blotting The dissolved protein can be stored at 20 C for several months or at 4 C for several days Genomic DNA purification 21 Add 500 pl Buffer AW1 to the AllPrep DNA spin column from step 5 Close the lid gently and centrifuge for 15 s at gt 8000 x g 10 000 rpm Discard the flow through Reuse the spin column in step 22 Note Buffer AW1 is supplied as a concentrate Ensure that ethanol is added to Buffer AW 1 before use see Things to do before starting 22 Add 500 pl Buffer AW2 to the AllPrep DN
48. luding any precipitate that may have formed to an RNeasy spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 15 s at gt 8000 x g gt 10 000 rpm Transfer the flow through to a 2 ml tube supplied for protein purification in steps 14 20 Reuse the collection tube in step 8 If the sample volume exceeds 700 pl centrifuge successive aliquots in the same RNeasy spin column Transfer the flow through after each centrifugation to the 2 ml tube Add 700 pl Buffer RW1 to the RNeasy spin column Close the lid gently and centrifuge for 15 s at gt 8000 x g 210 000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in step 9 Note After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Be sure to empty the collection tube completely Add 500 pl Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 15 s at gt 8000 x g 210 000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in step 10 Note Buffer RPE is supplied as a concentrate Ensure that ethanol is added to Buffer RPE before use see Things to do before starting Add 500 pl Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 2 min at gt 8000 x g 210 000 rpm to wash the spin column membrane The long centrifugat
49. mination No currently available purification method can guarantee that RNA is completely free of DNA even when it is not visible on an agarose gel While the the vast majority of cellular DNA will bind to the AllPrep DNA spin column trace amounts may still remain in the purified RNA depending on the amount and nature of the sample For analysis of very low abundance targets any interference by residual DNA contamination can be detected by performing realtime RT PCR control experiments in which no reverse transcriptase is added prior to the PCR step Wilfinger W W Mackey M and Chomczynski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity BioTechniques 22 474 Values up to 2 3 are routinely obtained for pure RNA in 10 mM Tris Cl pH 7 5 with some spectrophotometers AllPrep DNA RNA Protein Mini Handbook 09 2011 45 To prevent any interference by DNA in real time RT PCR applications such as with Applied Biosystems and LightCycler instruments we recommend designing primers that anneal at intron splice junctions so that genomic DNA will not be amplified QuantiTect Primer Assays from QIAGEN www giagen com GeneGlobe are designed for SYBR Green based real time RT PCR analysis of RNA sequences without detection of genomic DNA where possible For realtime RT PCR assays where amplification of genomic DNA cannot be avoided the QuantiTect Reverse Transcription Kit provi
50. n to dry the RNeasy spin column membrane if any flow through is present on the outside of the column step 11 of the protocols No protein detected on western blot or Coomassie stained gel Protein pellet does not completely dissolve a b 40 Protein pellet lost Protein pellet not soluble in the resuspension buffer used Protein pellet not completely solubilized In the protein precipitation procedure the protein pellet is only loosely attached to the side of the tube Be sure to decant the supernatant gently See also Protein pellet does not completely dissolve below Due to the different isoelectric points of proteins it is impossible to dissolve all proteins at a certain pH If your protein s of interest do not dissolve in the resuspension buffer change the pH In addition several types of protein are very difficult to solubilize especially membrane proteins To improve solubility use a different resuspension buffer containing other detergent s more suitable for your protein of interest The protein pellet may contain other cellular components that are insoluble in the resuspension buffer Resuspend disturb the pellet by pipetting up and down several times Then briefly centrifuge the sample and use the supernatant for downstream analysis AllPrep DNA RNA Protein Mini Handbook 09 2011 Comments and suggestions For greater solubilization of proteins dissolve the protein pellet in 5
51. ng a mortar and pestle freeze the tissue sample immediately in liquid nitrogen and grind to a fine powder under liquid nitrogen Transfer the suspension tissue powder and liquid nitrogen into a liquid nitrogen cooled appropriately sized tube and allow the liquid nitrogen to evaporate without allowing the sample to thaw Add lysis buffer and continue as quickly as possible with the homogenization according to one of the 2 methods below Note Grinding the sample using a mortar and pestle will disrupt the sample but will not homogenize it Homogenization must be performed afterwards Homogenization using QlAshredder homogenizers Using QlAshredder homogenizers is a fast and efficient way to homogenize cell and tissue lysates without cross contamination of samples Up to 700 pl of lysate is loaded onto a QlAshredder spin column placed in a 2 ml collection tube and spun for 2 minutes at maximum speed in a microcentrifuge The lysate is homogenized as it passes through the spin column QlAshredder homogenizers typically result in less DNA fragmentation compared with rotor stator homgenizers Homogenization using a syringe and needle Cell and tissue lysates can be homogenized using a syringe and needle Lysate is passed through a 20 gauge 0 9 mm needle attached to a sterile plastic syringe at least 5 10 times or until a homogeneous lysate is achieved Increasing the volume of lysis buffer may be required to facilitate handling and minimize loss
52. nol is added to the flow through from the AllPrep DNA spin column to provide appropriate binding conditions for RNA and the sample is then applied to an RNeasy spin column where total RNA binds to the membrane and contaminants are efficiently washed away High quality RNA is then eluted in 30 pl or more of water Buffer APP a novel aqueous protein precipitation solution is added to the flow through of the RNeasy spin column and the precipitated proteins are pelleted by centrifugation Intact total proteins are redissolved in an appropriate buffer and then ready to use in downstream applications The kit includes Buffer ALO which is compatible with SDS PAGE for dissolving the protein pellet In this handbook different protocols are provided for different starting materials The protocols differ primarily in the lysis and homogenization of the sample Once the sample is applied to the AllPrep DNA spin column the protocols are similar see flowchart next page Samples with particularly high DNA content may require additional DNase digestion AllPrep DNA RNA Protein Mini Handbook 09 2011 9 AllPrep DNA RNA Protein Procedure Cells or Tissue a DNA Bind DNA to Allprep column RNA and protein Add ethanol to flow through Bind RNA to RNeasy column RNA Wash RNeasy column eT Elute RNA Total RNA We recommend stabilizing harvested tissues immediately in Allprotect Tissue Reagent to protect DNA RNA
53. ons Enzyme 210210 Kit 25 Mix 5x PCR Buffer dNTP Mix 5x Q Solution RNase Free Water Related products for real time PCR and RT PCR applications QuantiTect Primer Assays for use in real time RT PCR with SYBR Green detection search for and order assays at www qiagen com GeneGlobe QuantiTect Primer Assay 200 For 200 x 50 pl reactions or Varies 400 x 25 pl reactions 10x QuantiTect Primer Assay lyophilized QuantiTect Reverse Transcription Kit for fast cDNA synthesis for sensitive real time two step RT PCR QuantiTect Reverse For 50 x 20 pl reactions gDNA 205311 Transcription Kit 50 Wipeout Buffer Quantiscript Reverse Transcriptase Quantiscript RT Buffer RT Primer Mix RNase Free Water Larger kit sizes also available see www qiagen com AllPrep DNA RNA Protein Mini Handbook 09 2011 57 Ordering Information Product Contents QuantiFast SYBR Green PCR Kit for fast quantitative real time PCR and two step RT PCR using SYBR Green QuantiFast SYBR Green For 400 x 25 pl reactions PCR Kit 400 3 x 1 7 ml 2x Master Mix contains ROX dye 2 x 2 ml RNase Free Water QuantiFast SYBR Green RT PCR Kit for fast quantitative real time one step RT PCR using SYBR Green QuantiFast SYBR Green For 400 x 25 pl reactions RT PCR Kit 400 3 x 1 7 ml 2x Master Mix contains ROX dye 100 pl RT Mix 2 x 2 ml RNase Free Water Cat no 204054 204154 For up to date licensing information an
54. operty to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the AllPrep DNA RNA Protein Mini Handbook and additional protocols available at www giagen com Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www giagen com 2007 2011 QIAGEN all rights reserved www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 5
55. ounts of a wide range of cultured cells and harvested tissues of animal and human origin The AllPrep DNA RNA Protein Mini Kit allows the parallel processing of multiple samples Methods involving the use of toxic substances such as phenol and or chloroform or time consuming and tedious methods such as alcohol precipitation are replaced by the AllPrep DNA RNA Protein procedure Genomic DNA purified with the AllPrep DNA RNA Protein procedure has an average length of 15 30 kb depending on homogenization conditions DNA of this length is particularly suitable for PCR where complete denaturation of the template is important to achieve the highest amplification efficiency The purified DNA is ready to use in any downstream application including M PCR and realtime PCR MH Southern dot and slot blot analyses E Comparative genome hybridization CGH E Genotyping SNP analysis With the AllPrep DNA RNA Protein procedure all RNA molecules longer than 200 nucleotides are purified The procedure provides an enrichment for mRNA since most RNAs lt 200 nucleotides such as 5 8S rRNA 5S rRNA and tRNAs which together comprise 15 20 of total RNA are selectively excluded The purified RNA is ready to use in any downstream application including E RT PCR Quantitative real time RT PCRt Differential display cDNA synthesis Northern dot and slot blot analyses Primer extension For purification of high molecular weight DNA we recomm
56. p 3 Note Ensure that B ME is added to Buffer RLT before use see Things to do before starting AllPrep DNA RNA Protein Mini Handbook 09 2011 21 Table 6 Volumes of Buffer RLT for direct cell lysis Dish diameter Volume of Buffer RLT lt 6 cm 350 pl 6 10 cm 600 pl Regardless of the cell number use the buffer volumes indicated to completely cover the surface of the dish 3a 3b 3c 22 Homogenize the lysate according to step 3a 3b or 3c See Disrupting and homogenizing starting material page 14 for more details on homogenization If processing lt 1 x 10 cells they can be homogenized by vortexing for 1 min After homogenization proceed to step 4 Note Incomplete homogenization leads to significantly reduced RNA yields and can cause clogging of the AllPrep DNA and RNeasy spin columns Homogenization with the TissueRuptor or QlAshredder homogenizer generally results in higher nucleic acid yields than with a syringe and needle Pipet the lysate directly into a QlAshredder spin column placed in a 2 ml collection tube and centrifuge for 2 min at full speed Proceed to step 4 Place the tip of the TissueRuptor disposable probe into the lysate and operate the TissueRuptor at full speed until the lysate is homogenous usually 30 s Proceed to step 4 Note To avoid damage to the TissueRuptor and disposable probe during operation make sure the tip of the probe remains submerged in the b
57. p DNA spin column Try using smaller samples Alternatively perform DNase digestion on the RNeasy spin column membrane see Appendix E page 51 or perform DNase digestion of the eluted RNA followed by RNA cleanup Use 10 mM Tris Cl pH 7 5 not RNase free water to dilute the sample before measuring purity see Appendix B page 44 Ensure that tissue samples are properly stabilized and stored in Allprotect Tissue Reagent or RNAlater RNA Stabilization Reagent For frozen cell pellets or frozen tissue samples ensure that they were flash frozen immediately in liquid nitrogen and properly stored at 70 C Perform the AllPrep DNA RNA Protein procedure quickly especially the first few steps See Appendix A page 42 and Handling and storing starting material page 14 When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 38 AllPrep DNA RNA Protein Mini Handbook 09 2011 Comments and suggestions b RNase contamination Although all AllPrep buffers have been tested and are guaranteed RNase free RNases can be introduced during use Be certain not to introduce any RNases during the AllPrep DNA RNA Protein procedure or later handling See Appendix A page 42 for general remarks on handling RNA DNA fragmented Homogenization too vigorous The
58. ratory manual 2nd ed Cold Spring Harbor NY Cold Spring Harbor Laboratory Press FA gel preparation To prepare FA gel 1 2 agarose of size 10 x 14 x 0 7 cm mix 1 2 g agarose 10 ml 10x FA gel buffer see composition below Add RNase free water to 100 ml If smaller or larger gels are needed adjust the quantities of components proportionately Heat the mixture to melt agarose Cool to 65 C in a water bath Add 1 8 ml of 37 12 3 M formaldehyde and 1 pl of a 10 mg ml ethidium bromide stock solution Mix thoroughly and pour onto gel support Prior to running the gel equilibrate in 1x FA gel running buffer see composition below for at least 30 minutes RNA sample preparation for FA gel electrophoresis Add 1 volume of 5x RNA loading buffer see composition below to 4 volumes of RNA sample for example 10 pl of loading buffer and 40 pl of RNA and mix Incubate for 3 5 minutes at 65 C chill on ice and load onto the equilibrated FA gel Gel running conditions Run gel at 5 7 V cm in 1x FA gel running buffer When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier AllPrep DNA RNA Protein Mini Handbook 09 2011 47 Composition of FA gel buffers 10x FA gel buffer 200 mM 3 N morpholino propanesulfonic acid MOPS free acid 50 mM sodium acet
59. rial safety data sheets MSDSs These are available online in convenient and compact PDF format at www qiagen com Support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste Buffer AW1 contains guanidine hydrochloride Buffer RLT contains guanidine thiocyanate and Buffer RW1 contains a small amount of guanidine thiocyanate Guanidine salts can form highly reactive compounds when combined with bleach If liquid containing these buffers is spilt clean with suitable laboratory detergent and water If the spilt liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite The following risk and safety phrases apply to the components of the AllPrep DNA RNA Protein Mini Kit Buffer AW1 Contains guanidine hydrochloride harmful irritant Risk and safety phrases R22 36 38 S13 26 36 46 Buffer RLT Contains guanidine thiocyanate harmful Risk and safety phrases R20 21 22 32 13 26 36 46 Buffer RW1 Contains ethanol flammable Risk phrase R10 Buffer APP Contains zinc chloride dangerous for the environment Risk phrase R52 53 R10 Flammable R20 21 22 Harmful by inhalation in contact with skin and if swallowed R22 Harmful if swallowed R32 Contact with acids liberates very toxic gas R36 38
60. rious culture vessels Cell culture vessel Growth area cm Number of cells Multiwell plates E 96 well 0 32 0 6 4 5 x 104 E 48 well 1 OF E 24 well 2 2 5 x 105 E 12 well 4 5x 10 E 6 well 9 5 1x 10 Dishes E 35mm 8 1 x 10 60mm 21 Phe 5 IOP mS 100mm 56 7 x 10 a 145 150 mm 145 2 xan Flasks mM 40 50 nl 25 3 x 10 M 250 300 ml 75 TEO E 650 750 ml 162 175 2x10 Per well if multiwell plates are used varies slightly depending on the supplier t Cell numbers are given for Hela cells approximate length 15 pm assuming confluent growth Cell numbers will vary for different kinds of animal and human cells which vary in length from 10 to 30 pm Important points before starting If using the AllPrep DNA RNA Protein Mini Kit for the first time read Important Notes page 12 If preparing RNA for the first time read Appendix A page 42 If using the TissueRuptor ensure that you are familiar with operating it by referring to the TissueRuptor User Manual and TissueRuptor Handbook Cell pellets can be stored at 70 C for later use or used directly in the procedure Determine the number of cells before freezing Frozen cell pellets should be thawed slightly so that they can be dislodged by flicking the tube in step 2 Homogenized cell lysates from step 3 can be stored at 70 C for several months Frozen lysates should be incubated at 37 C in a water bath until completely thawed and salts are
61. room temperature 15 25 C or at 4 C for later DNA purification in steps 21 24 Use the flow through for RNA purification in steps 6 13 Note Do not store the AllPrep DNA spin column at room temperature or at 4 C for long periods Do not freeze the column Total RNA purification 6 To the flow through from step 5 add 96 100 ethanol either 250 pl if 350 pl Buffer RLT was used or 430 pl if 600 pl Buffer RLT was used Mix well by pipetting Do not centrifuge Proceed immediately to step 7 If some lysate was lost during homogenization and DNA binding to the AllPrep DNA spin column adjust the volume of ethanol accordingly Note Precipitates may be visible after addition of ethanol but this does not affect the procedure AllPrep DNA RNA Protein Mini Handbook 09 2011 31 Transfer up to 700 pl of the sample including any precipitate that may have formed to an RNeasy spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 15 s at gt 8000 x g gt 10 000 rpm Transfer the flow through to a 2 ml tube supplied for protein purification in steps 14 20 Reuse the collection tube in step 8 If the sample volume exceeds 700 yl centrifuge successive aliquots in the same RNeasy spin column Transfer the flow through after each centrifugation to the 2 ml tube Add 700 pl Buffer RW1 to the RNeasy spin column Close the lid gently and centrifuge for 15 s at gt 8000 x g 210 000 rpm
62. ssues and from cells yeast bacteria or viruses DNeasy Blood amp Tissue 50 DNeasy Mini Spin Columns 69504 Kit 50 Proteinase K Buffers Collection Tubes 2 ml QIAGEN Genomic tips for purification of high molecular weight DNA from a wide range of samples QIAGEN Genomic tip 20 G 25 columns maximum DNA binding 10223 capacity of 20 pg Genomic DNA Buffer Set Buffers including specific lysis 19060 buffers for yeast bacteria cells blood and tissue Larger kit sizes and or formats available see www qiagen com AllPrep DNA RNA Protein Mini Handbook 09 2011 55 Ordering Information Product Contents Cat no Blood amp Cell Culture DNA Kits for purification of high molecular weight DNA from blood and cultured cells Blood amp Cell Culture DNA 25 QIAGEN Genomic tip 20 G 13323 Mini Kit 25 QIAGEN Protease Buffers Related products for total RNA purification RNAlater RNA Stabilization Reagent for immediate stabilization of the gene expression profile in harvested tissues RNAlater RNA Stabilization For stabilization of RNA in 76104 Reagent 50 ml 25 x 200 mg tissue samples 50 ml RNAlater RNA Stabilization Reagent RNAlater RNA Stabilization For stabilization of RNA in 76106 Reagent 250 ml 125 x 200 mg tissue samples 250 ml RNAlater RNA Stabilization Reagent RNeasy Mini Kit for purification of up to 100 pg total RNA from animal cells or tissues RNeasy Mini Kit 50 50 RNeasy Mini Spin Columns 741
63. stable for at least 6 months under these conditions 4 AllPrep DNA RNA Protein Mini Handbook 09 2011 Product Use Limitations The AllPrep DNA RNA Protein Mini Kit is intended for molecular biology applications This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the product We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on
64. strength on the spectrophotometric assessment of nucleic acid purity BioTechniques 22 474 t When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 50 AllPrep DNA RNA Protein Mini Handbook 09 2011 Appendix E Optional On Column DNase Digestion Using the RNase Free DNase Set Although DNA binds very efficiently to the AllPrep DNA spin column some tissues contain very high amounts of DNA e g spleen and thymus and will overload the AllPrep DNA spin column unless the amount of starting material is very small For these tissues we recommend performing DNase digestion on the RNeasy spin column membrane if the eluted RNA will be used in downstream applications sensitive to very small amounts of DNA Tissues containing moderate amounts of DNA and cultured cells do not require DNase digestion The QIAGEN RNase Free DNase Set see page 54 for ordering information provides efficient on column digestion of DNA during RNA purification The DNase is efficiently removed in subsequent wash steps Note Standard DNase buffers are not compatible with on column DNase digestion Using other buffers may affect the binding of the RNA to the RNeasy spin column membrane reducing the yield and integrity of the RNA Preparation of tissue homogenates and binding of RNA to the RNeasy spin
65. tarting material in order to obtain optimal nucleic acid yield and purity The maximum amount that can be used is limited by The type of sample and its DNA and RNA content MH The volume of Buffer RLT required for efficient lysis E The DNA binding capacity of the AllPrep DNA spin column E The RNA binding capacity of the RNeasy spin column When processing samples containing high amounts of DNA or RNA less than the maximum amount of starting material shown in Table 1 should be used so that the binding capacity of the spin columns are not exceeded When processing samples containing average or low amounts of DNA and RNA the maximum amount of starting material shown in Table 1 can be used However even though the binding capacity of the spin columns are not reached the maximum amount of starting material must not be exceeded Otherwise lysis will be incomplete and cellular debris may interfere with the binding of nucleic acids to the spin column membranes resulting in lower yield and purity of DNA and RNA More information on using the correct amount of starting material is given in each protocol Table 2 shows expected DNA and RNA yields from various cells and tissues Note Although the AllPrep DNA spin column can bind a maximum of 100 yg DNA the use of starting materials containing more than 20 pg DNA may lead to the purification of RNA containing small amounts of DNA If the binding capacity of the RNeasy spin column is exceeded RNA yi
66. the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www qiagen com Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If you have any questions or experience any difficulties regarding the AllPrep DNA RNA Protein Mini Kit or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www qiagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com AllPrep DNA RNA Protein Mini Handbook 09 2011 5 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate mate
67. the final RNA concentration will be higher Total protein precipitation 14 15 16 Add 1 volume usually 600 or 1000 pl of Buffer APP to the flow through from step 7 Mix vigorously and incubate at room temperature for 10 min to precipitate protein Centrifuge at full speed for 10 min and carefully decant the supernatant Add 500 pl of 70 ethanol to the protein pellet Centrifuge at full speed for 1 min and remove the supernatant by using a pipet or by decanting as much liquid as possible It is not necessary to resuspend or incubate the pellet Dry the protein pellet for 5 10 min at room temperature Note Incomplete drying may cause problems when loading the protein onto a gel due to residual ethanol Add up to 100 pl Buffer ALO and mix vigorously to dissolve the protein pellet Note Ensure that DTT is added to Buffer ALO before use see Things to do before starting The volume of Buffer ALO to add depends on the amount of starting material See Table 2 page 13 for typical protein yields from various starting materials Supernatant contains Buffer RLT and is therefore not compatible with bleach See page 6 for safety information 24 AllPrep DNA RNA Protein Mini Handbook 09 2011 Buffer ALO is a Laemmli related sample buffer for use in SDS PAGE and contains bromophenol blue dye If the proteins will not be analyzed by SDS PAGE dissolve the pellet in a buffer compatible with the intended downstream
68. the precipitated protein is highly denatured and shows reduced solubility in water Resolubilization of the protein is possible in Buffer ALO or SDS PAGE sample buffer or in other solutions such as 5 w v SDS or 8 M urea Protein dissolved in SDS or urea solutions can be quantified using the BCA bicinchoninic acid method but there must be no dye in the solutions Protein in 5 w v SDS can be used directly with the method but protein dissolved in 8 M urea must be diluted to give 3 M urea AllPrep DNA RNA Protein Mini Handbook 09 2011 53 Ordering Information Product Contents Cat no AllPrep DNA RNA 50 AllPrep DNA Mini Spin Columns 80004 Protein Mini Kit 50 50 RNeasy Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers Accessories Collection Tubes 2 ml 1000 x 2 ml Collection Tubes 19201 Allprotect Tissue 100 ml Allprotect Tissue Reagent 76405 Reagent 100 ml Allprotect Reagent Pump QlAshredder 50 50 disposable cell lysate 79654 homogenizers QlAshredder 250 250 disposable cell lysate 79656 homogenizers TissueRuptor Handheld rotor stator homogenizer Varies 5 TissueRuptor Disposable Probes TissueRuptor Disposable 25 nonsterile plastic disposable 990890 Probes 25 probes for use with the TissueRuptor TissueLyser Universal laboratory mixer mill Varies disruptor TissueLyser Adapter Set 2 x 24 2 sets of Adapter Plates and 2 racks 69982 for use with 2 ml microcentrifuge tubes on the TissueL
69. to 25 C Warm the lysate to 37 C before transferring it to the AllPrep DNA spin column Low nucleic acid yield a Insufficient disruption and See Disrupting and homogenizing starting homogenization material page 14 for details on disruption and homogenization methods In subsequent preparations reduce the amount of starting material see protocols pages 18 and 27 and or increase the volume of lysis buffer and the homogenization time 36 AllPrep DNA RNA Protein Mini Handbook 09 2011 Comments and suggestions b Too much starting material c RNA still bound to RNeasy spin column membrane d DNA still bound to AllPrep DNA spin column membrane e Ethanol carryover f Incomplete removal of cell culture medium cell samples DNA contaminated with RNA a Lysate applied to the AllPrep DNA spin column contains ethanol b Sample is affecting pH of homogenate Overloading the spin columns significantly reduces nucleic acid yields Reduce the amount of starting material see page 12 Repeat RNA elution but incubate the RNeasy spin column on the benchtop for 10 min with RNase free water before centrifuging Repeat DNA elution but incubate the AllPrep DNA spin column on the benchtop for 10 min with Buffer EB before centrifuging During the second wash with Buffer RPE be sure to centrifuge at 28000 x g 210 000 rpm for 2 min at 20 25 C to dry the RNeasy spin column membrane Perform the option
70. tream applications that require long DNA fragments Note Rotor stator homogenization using a metal probe must be avoided as the probe can warm the homogenate resulting in protein degradation To preserve intact proteins rotor stator homogenization should be carried out with the TissueRuptor which uses a plastic disposable probe AllPrep DNA RNA Protein Mini Handbook 09 2011 15 Disruption and homogenization using the TissueLyser In bead milling tissues can be disrupted by rapid agitation in the presence of beads and lysis buffer Disruption and simultaneous homogenization occur by the shearing and crushing action of the beads as they collide with the cells The Tissuelyser disrupts and homogenizes up to 48 tissue samples simultaneously when used in combination with the Tissuelyser Adapter Set 2 x 24 which holds 48 x 2 ml microcentrifuge tubes containing stainless steel beads of 5 mm mean diameter For guidelines on using the TissueLyser refer to the Tissuelyser Handbook For other bead mills refer to suppliers guidelines Note Tungsten carbide beads react with Buffer RLT and must not be used to disrupt and homogenize tissues The Tissuelyser can also disrupt and homogenize up to 192 tissue samples simultaneously when used in combination with the TissueLyser Adapter Set 2 x 96 which holds 192 x 1 2 ml microtubes containing stainless steel beads of 5 mm mean diameter Disruption using a mortar and pestle For disruption usi
71. uffer Pass the lysate at least 5 times through a blunt 20 gauge needle 0 9 mm diameter fitted to an RNase free syringe Proceed to step 4 Transfer the homogenized lysate to an AllPrep DNA spin column placed in a 2 ml collection tube supplied Close the lid gently and centrifuge for 30 s at gt 8000 x g 210 000 rpm Note Make sure that no liquid remains on the column membrane after centrifugation If necessary repeat the centrifugation until all liquid has passed through the membrane Place the AllPrep DNA spin column in a new 2 ml collection tube supplied and store at room temperature 15 25 C or at 4 C for later DNA purification in steps 21 24 Use the flow through for RNA purification in steps 6 13 Note Do not store the AllPrep DNA spin column at room temperature or at 4 C for long periods Do not freeze the column AllPrep DNA RNA Protein Mini Handbook 09 2011 Total RNA purification 6 To the flow through from step 5 add 96 100 ethanol either 250 pl if 350 pl Buffer RLT was used or 400 pl if 600 pl Buffer RLT was used Mix well by pipetting Do not centrifuge Proceed immediately to step 7 If some lysate was lost during homogenization and DNA binding to the AllPrep DNA spin column adjust the volume of ethanol accordingly Note When purifying RNA from certain cell lines precipitates may be visible after addition of ethanol This does not affect the procedure Transfer up to 700 pl of the sample inc
72. ways wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier 44 AllPrep DNA RNA Protein Mini Handbook 09 2011 Concentration of RNA sample 44 pg ml x Azo x dilution factor 44 pg ml x 0 2 x 50 440 pg ml Total amount concentration x volume in milliliters 440 pg ml x 0 1 ml 44 pg of RNA Purity of RNA The ratio of the readings at 260 nm and 280 nm Az60 Aogo provides an estimate of the purity of RNA with respect to contaminants that absorb in the UV spectrum such as protein However the Aygo Aygo ratio is influenced considerably by pH Since water is not buffered the pH and the resulting Agso Avgo ratio can vary greatly Lower pH results in a lower Apggo Aogo ratio and reduced sensitivity to protein contamination For accurate values we recommend measuring absorbance in 10 mM Tris Cl pH 7 5 Pure RNA has an Aogo Azgo ratio of 1 9 2 1 in 10 mM Tris Cl pH 7 5 Always be sure to calibrate the spectrophotometer with the same solution used for dilution For determination of RNA concentration however we recommend dilution of the sample in a buffer with neutral pH since the relationship between absorbance and concentration Ajo reading of 1 44 pg ml RNA is based on an extinction coefficient calculated for RNA at neutral pH see Spectrophotometric quantification of RNA page 44 DNA conta
73. y into ethanol and CO When preparing Tris buffers treat water with DEPC first and then dissolve Tris to make the appropriate buffer Trace amounts of DEPC will modify purine residues in RNA by carbethoxylation Carbethoxylated RNA is translated with very low efficiency in cell free systems However its ability to form DNA RNA or RNA RNA hybrids is not seriously affected unless a large fraction of the purine residues have been modified Residual DEPC must always be eliminated from solutions or vessels by autoclaving or heating to 100 C for 15 minutes Note AllPrep buffers are guaranteed RNase free without using DEPC treatment and are therefore free of any DEPC contamination When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Plastics used for some electrophoresis tanks are not resistant to ethanol Take proper care and check the supplier s instructions AllPrep DNA RNA Protein Mini Handbook 09 2011 43 Appendix B Storage Quantification and Determination of Quality of RNA Storage of RNA Purified RNA may be stored at 20 C or 70 C in RNase free water Under these conditions no degradation of RNA is detectable after 1 year Quantification of RNA The concentration of RNA should be determined by measuring the absorbance at 260 nm Aso in a spectrophotometer
74. yser Stainless Steel Beads Stainless Steel Beads suitable for use 69989 5 mm 200 with the TissueLyser system TissueLyser Single Bead For dispensing individual beads 69965 Dispenser 5 mm 5 mm diameter RNase Free DNase Set 50 For 50 RNA minipreps DNase I 79254 Buffer RDD and Water all RNase Free Visit www giagen com automation to find out more about the TissueRuptor and TissueLyser and to order 54 AllPrep DNA RNA Protein Mini Handbook 09 2011 Ordering Information Product Contents Cat no Related products for sample preparation for systems biology AllPrep DNA RNA Kits for simultaneous purification of genomic DNA and total RNA from the same cell or tissue sample AllPrep DNA RNA Micro 50 AllPrep DNA Mini Spin Columns 80284 Kit 50 50 RNeasy MinElute Spin Columns Collection Tubes Carrier RNA RNase Free Reagents and Buffers AllPrep DNA RNA Mini 50 AllPrep DNA Mini Spin Columns 80204 Kit 50 50 RNeasy Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers AllPrep RNA Protein Kit for simultaneous purification of total RNA and native protein from the same cultured cell sample AllPrep RNA Protein Kit 50 50 AllPrep Mini Spin Columns 80404 50 RNeasy Mini Spin Columns 50 Protein Cleanup Mini Spin Columns Collection Tubes RNase Free Reagents and Buffers Related products for genomic DNA purification DNeasy Blood amp Tissue Kit for purification of total DNA from animal blood and ti

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AllPrep® DNA/RNA/Protein Mini Handbook