SuperScript III Platinum CellsDirect Two-Step qRT
Contents
1. For adherent cells grown in tissue culture wells i e in 24 well 48 well or 96 well plates perform the following lysis procedure 1 Aspirate the media in each well and wash each well with 1X cold PBS Aspirate the PBS 2 Add the Lysis Enhancer Resuspension Buffer master mix see Note above to each well For 96 well plates add at least 11 ul of the buffer enhancer mix to each well For 24 well plates add at least 110 ul of the buffer enhancer mix to each well The master mix should cover the cells in the well 3 Incubate the plates on ice for up to 10 minutes During that period tap the plate periodically and check the cells under a microscope every 2 3 minutes to see whether they have detached or burst 4 After 10 minutes gently pipet the cells up and down to dislodge the remaining attached cells 5 Transfer 10 ul of the cell suspension to a 0 2 ml thin walled PCR tube or plate well Control Reaction For the control reaction add 10 ul of Resuspension Buffer and 1 ul of Lysis Enhancer to a PCR tube or plate well and then add 1 ul of Control HeLa Total RNA 6 Transfer the tube plate to an incubator or thermal cycler preheated to 75 C and incubate for 10 minutes Control Reaction For the control reaction incubate for 3 minutes 7 After incubation spin briefly to collect the condensation and proceed to DNase I Digestion page 7 Continued on next page Lysing Cells continued DNase In this step y2. 10 11 12 13 14 15 Add enough trypsin to cover the adherent cells in your tissue culture dish plate or flask e g for a 10 cm dish use 1 ml for a T75 flask use 3 ml Incubate for 5 minutes at room temperate or in a 37 C incubator Check for cell detachment under a microscope If cells have not detached gently tap the disk or flask to dislodge the cells or let the cells incubate longer checking them every minute under a microscope When all the cells have detached add serum containing media to a final volume of 10 ml for 6 and 12 well plates add a 1X 2X volume of media Note that the media must contain serum to inactivate the trypsin Pipet the cells gently up and down to mix and then transfer the cell suspension to a centrifuge tube Spin the cells at 200 x g for 5 minutes to pellet or spin at the recommended speed and time for your cell line Aspirate the media and wash the cell pellet with 5 10 ml of 1X cold PBS Spin the cells at 200 x g for 5 minutes to pellet Aspirate the PBS and resuspend the pellet in 500 ul to 1 ml of 1X cold PBS Mix the cell solution gently Collect a small aliquot to verify that the cells are at the desired concentration Determine cell density electronically using a Coulter Counter or manually using a hemacytometer chamber Adjust the cell density using cold PBS so that it falls within the range of 1 10 000 cells ul Count the cells again to verify cell concentrat
3. Prepare a master mix of all components except template as specified on the previous page For each reaction add 42 49 ul of the master mix depending on template volume to a 0 2 ml microcentrifuge tube or each well of a 96 well PCR plate Add 1 8 ul of cDNA template from the first strand synthesis reaction Step 7 page 8 to each reaction vessel for a final reaction volume of 50 ul Use 4 ul of template as a general starting point Cap or seal the tube plate Gently mix and make sure that all components are at the bottom of the reaction vessel Centrifuge briefly if needed Place reactions in a thermal cycler programmed as described above After cycling hold the reaction at 4 C until further analysis Collect and analyze the results qPCR Roche LightCycler Introduction Cycling Program Master Mix This section provides a cycling program reaction mixture and protocol for the Roche LightCycler After programming the instrument and preparing the reaction mix follow the protocol on the following page to perform the reaction Program the LightCycler as follows Program choice Amplification Analysis mode Quantification 50 C for 2 minutes hold UDG incubation 92 C for 1 minute hold 50 cycles of 92 C 5 seconds 60 C 30 seconds single acquire Melting Curve Analysis Program choice Melting curve Analysis mode Melting curves 95 C 0 seconds 20 C second transition 55 C 15 seco
4. identify the presence of primer dimers and analyze the specificity of the reaction Melting curve analysis can identify primer dimers by their lower annealing temperature compared to that of the amplicon The presence of primer dimers in samples containing template decreases PCR efficiency and obscures analysis and determination of cycle thresholds The formation of primer dimers most often occurs in no template controls where the polymerase enzyme is essentially idle and in this case the quantitative analysis of the template samples is not affected Melting curve analysis of no template controls can discriminate between primer dimers and spurious amplification due to contaminating nucleic acids in reagent components Platinum SYBR Green qPCR SuperMix UDG includes magnesium chloride at a final concentration of 3 mM Optimal performance for any given target may require adjusting this level of magnesium If necessary use the 50 mM magnesium chloride solution included in the kit to increase the magnesium concentration ROX Reference Dye can be used to adjust for non PCR related fluctuations in fluorescence between reactions and provides a stable baseline in multiplex reactions Its use is optional It is composed of a glycine conjugate of 5 carboxy X rhodamine succinimidyl ester 25 uM in 20 mM Tris HCl pH 8 4 0 1 mM EDTA and 0 01 Tween 20 ROX is supplied at 50X concentration Add 1 ul of ROX for every 50 ul of reaction volu
5. 1 ul of sterile distilled water and 1 ul of RNase OUT Recombinant Ribonuclease Inhibitor instead of the RT Enzyme Mix Spin the tube plate briefly to collect the contents Transfer the tube plate to a thermal cycler preheated to 25 C and incubate for 10 minutes Incubate at 50 C for 20 minutes Inactivate the reaction at 85 C for 5 minutes Add 1 ul of RNase H 2 U pl to each tube well and incubate at 37 C for 20 minutes Chill the reaction on ice and store at 20 C or proceed directly to qPCR qPCR Guidelines and Recommendations Introduction Required Materials Instrument Settings Primers Amplicon Size Note After first strand cDNA synthesis you can proceed directly to qPCR without additional purification The following materials are provided by the user e qPCR instrument e Appropriate PCR plates tubes for instrument e Primers e Pipettes The following materials are provided in the kit e Components of the qPCR module Platinum SYBR Green qPCR SuperMix UDG can be used with a variety of instruments including the ABI PRISM 7000 7300 7500 7700 7900 and GeneAmp 5700 Bio Rad iCycler Stratagene Mx4000 and Mx3000P Corbett Research Rotor Gene MJ Research DNA Engine Opticon and Opticon 2 Cepheid Smart Cycler and Roche LightCycler Optimal cycling conditions will vary refer to your instrument manual for operating instructions The protocols on the following p
6. Guide to Methods and Applications Innis M Gelfand D Sninsky J and White T eds p 46 Academic Press Inc Lindahl T Ljungquist S Siegert W Nyberg B and Sperens B 1977 DNA N glycosidases properties of uracil DNA glycosidase from Escherichia coli J Biol Chem 252 3286 Longo M Berninger M and Hartley J 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 Meateo R Sico E Wang L F Fung J 1994 Uracil DNA Glycosylase Minimizing Residual Enzyme Activity After Nested PCR Focus 16 4 104 5 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press Cold Spring Harbor Sharkey D J Scalice E R Christy K G Atwood S M and Daiss J L 1994 Antibodies as thermolabile switches high temperature triggering for the polymerase chain reaction BioTechnology 12 506 509 Simms D Cizdziel P E and Chomczynski P 1993 Focus 15 99 Wittwer C T Herrmann M G Moss A A and Rasmussen R P 1997 Continuous fluorescence monitoring of rapid cycle DNA amplification BioTechniques 22 130 138 Takagi M Nishioka M Kakihara H Kitabayashi M Inoue H Kawakami B Oka M and Imanaka T 1997 Appl Environ Microbiol 63 4504 Schwabe W Lee J E Nathan M Xu R H Sitaraman K Smith M Potter R J Rosenthal K Rashtchia
7. a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 or e mail outlicensing lifetech com Continued on next page 19 Purchaser Notification continued Limited Use Label License No 14 Direct Inhibition by An
8. ages have been optimized for the ABI PRISM 7700 and the Roche LightCycler Primer selection is one of the most important parameters for qPCR when using a SYBR Green detection system To design primers we strongly recommend using a primer design software program such as OligoPerfect available on the Web at www invitrogen com oligos In OligoPerfect designer enter your target sequence and select PCR Detection from the Application pulldown menu Using primer design software will ensure that primers are specific for the target sequence and free of internal secondary structure and avoid complementation at 3 ends within each primer and with each other When designing primers keep in mind that the amplicon length should be approximately 80 250 bp to optimize the efficiency of qPCR Optimal results may require a titration of primer concentrations between 100 and 500 nM A final concentration of 200 nM per primer is effective for most reactions For best results the amplicon should be limited to 80 250 bp in size Since PCR is a powerful technique capable of amplifying trace amounts of DNA all appropriate precautions should be taken to avoid cross contamination Continued on next page 9 qPCR Guidelines and Recommendations continued Melting Curve Analysis Magnesium Concentration ROX Reference Dye Bovine Serum Albumin BSA 10 Melting curve analysis should always be performed during qPCR to
9. and cDNA directly from total RNA The concentration of SuperScript III RT in this system has been optimized to synthesize first strand cDNA from total RNA in cell lysate Continued on next page Introduction Continued Platinum SYBR Green qPCR SuperMix UDG Control RNA Platinum SYBR Green qPCR SuperMix UDG is a ready to use reaction cocktail containing all components except primers for the amplification and detection of DNA in qPCR It contains SYBR Green I fluorescent dye Platinum Tag DNA polymerase Mg uracil DNA glycosylase UDG proprietary stabilizers and deoxyribonucleotide triphosphates dNTPs with dUTP instead of dTTP The concentration of the SuperMix allows for the addition of primers and template SYBR Green I is a fluorescent dye that binds directly to double stranded DNA dsDNA In qPCR as dsDNA accumulates the dye generates a signal that is proportional to the DNA concentration and that can be detected using qPCR instruments SYBR Green I in this SuperMix formulation can quantify as few as 10 copies of a target gene in as little as 1 pg of template DNA or RNA It has a broad dynamic range of six orders of magnitude and is compatible with melting curve analysis Platinum Taq DNA polymerase is precomplexed with specific monoclonal antibodies that inhibit polymerase activity during reaction assembly at room temperature Full polymerase activity is restored after the denaturation step in PCR
10. cycling providing an automatic hot start in PCR and thereby increasing amplification efficiency sensitivity and yield UDG and dUTP are included in the mixture to prevent the reamplification of carryover PCR products between reactions UTP in the mix ensures that any amplified DNA will contain uracil UDG or uracil N glycosylase removes uracil residues from single or double stranded DNA preventing dU containing DNA from serving as template in future PCRs Incubation of subsequent PCRs with UDG before cycling destroys any contaminating dU containing PCR product from previous reactions After this decontamination step UDG is inactivated by the high temperatures during normal PCR cycling thereby allowing the amplification of genuine target sequence s HeLa Total RNA is included in the kit as a control The concentration of HeLa Total RNA provided 10 ng ul is equivalent to 1 000 cells Lysing Cells Introduction Cell Types and Density Important Required Materials Note Methods In this step you lyse your cells in Resuspension Buffer and Lysis Enhancer and perform a DNase I digestion to remove genomic DNA from the sample This kit has been optimized for small cell samples ranging from 1 to 10 000 cells This kit is compatible with several different mammalian cell lines including HeLa COS 7 293 Jurkat CV1 and K562 Cells may be grown under a variety of conditions and treatments Any type of c
11. es Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners 23 Notes invitrogen by Life technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
12. f products were derived from DNA prepare a negative RT control Nonspecific Vary the annealing conditions annealing of qPCR F Optimize magnesium concentration for each primers template and primer combination 16 References Berger S L and Kimmel A R 1987 Methods Enzymol 152 316 Chomczynski P and Sacchi N 1987 Anal Biochem 162 156 Chou Q Russel M Birch D Raymond J and Bloch W 1992 Prevention of pre PCR mis priming and primer dimerization improves low copy number amplifications Nucl Acids Res 20 1717 Compton T 1990 in PCR Protocols A Guide to Methods and Applications Innis M Gelfand D Sninsky J and White T eds p 39 Academic Press Inc D Alessio J M Gruber C E Cain C and Noon M C 1990 Focus 12 47 Frohman M A Dush M K and Martin G R 1988 Proc Nat Acad Sci USA 85 8998 Gerard G F 1994 Focus 16 102 Gerard G F Schmidt B J Kotewicz M L and Campbell J H 1992 Focus 14 91 Higuchi R Fockler C Walsh P S Griffith R 1992 Simultaneous amplification and detection of specific DNA sequences Bio Technology 10 413 417 Ishiguro T Saitoh J Yawata H Yamagishi H Iwasaki S and Mitoma Y 1995 Homogeneous quantitative assay of hepatitis C virus RNA by polymerase chain reaction in the presence of a fluorescent intercalater Anal Biochem 229 207 Lee C C and Caskey T 1990 in PCR Protocols A
13. invitrogen by technologies SuperScript III Platinum CellsDirect Two Step qRT PCR Kit with SYBR Green For two step real time quantitative RT PCR from cell lysate using SYBR Green I fluorescent dye Catalog Nos 11738 060 and 11738 068 Rev Date 28 June 2010 Manual part no 250751 MAN0000472 ii Table of Contents Kit Contents and Storage Introduction ix RANG RO REOR t e A E eto e ene TY SiG Cell ter eR E e OR ente 4 First Strand cDNA Synthesis 8 qPCR Guidelines and Recommendations 4 9 qPCR Instruments Using PCR Tubes Plates sse 11 qPCR Roche EightCyeler 2 det e tibt a eve teles des 13 Troubleshooting tentent nnne R 15 References Related Prod cts 0 m REC eg Ee tad 18 Purchaser Nod nte RR 19 Technical Service e Re ERR A eco I n Dite eel 22 iii iv Kit Contents and Storage Shipping and Storage Kit Size and Modules cDNA Synthesis Module qPCR Module Kit components are shipped on dry ice and should be stored at 20 C ROX Reference Dye should be stored in the dark Each kit includes a cDNA Synthesis module and a qPCR module Each cDNA synthesis reaction provides enough cDNA for multiple qPCR reactions Number of Reactions Kit Catalog Number cDNA Synthesis gPCR 11737 060 25 100 11737 068 100 500 Component 100 rxn kit 500 rxn kit Resuspension Buffer 2 x 250 ul 2x1ml DNase I 1 U l 125 ul 500 p
14. ion To a 0 2 ml thin walled PCR tube or plate well on ice add 1 ul of Lysis Enhancer and 10 ul of Resuspension Buffer Note A master mix of Lysis Enhancer and Resuspension Buffer may be prepared for multiple reactions Transfer 1 2 ul of cells 10 000 cells to the PCR tube well Control Reaction For the control reaction add 1 ul of Control HeLa Total RNA to the PCR tube or plate well instead of cell lysate Transfer the tube plate to an incubator water bath or thermal cycler preheated to 75 C and incubate for 10 minutes Control Reaction For the control reaction incubate for 3 minutes After incubation spin briefly to collect the condensation and proceed to DNase I Digestion page 7 Continued on next page 5 Lysing Cells continued Note Important Lysing Cells in Tissue Culture Wells For adherent cells grown in tissue culture wells note the following e Seed cells in tissue culture wells so that 10 ul of resuspended cells will yield the desired concentration e Master mix Before starting the following procedure prepare a master mix of Lysis Enhancer and Resuspension buffer for multiple reactions Add 1 ul of Lysis Enhancer for every 10 ul of Resuspension Buffer You can order additional CellsDirect Resuspension Buffer and Lysis Enhancer from Invitrogen Catalog no 11739 010 Additional buffer and enhancer may be required if you are using 48 well or 24 well plates in your experiments
15. l 10X DNase I Buffer 40 ul 160 ul 25 mM EDTA 120 ul 400 ul RT Enzyme Mix contains SuperScript III RT 100 units ul and RNaseOUT Recombinant Ribonuclease Inhibitor 20 units ul 50 ul 200 ul 2X RT Reaction Mix 500 pl 2x1ml E Coli RNase H 2 U pl 30 ul 100 ul HeLa Total RNA 10 ng l 10 ul 10 pl Lysis Enhancer 125 ul 500 ul Oligo dT 2 5 uM random hexamers 2 5 ng ul 10 mM MgCb and dNTPs Component 100 rxn kit 500 rxn kit Platinum SYBR Green qPCR SuperMix UDG 2x125ml 125ml 50 mM Magnesium Chloride MgCl 1ml 2x1ml 20X Bovine Serum Albumin BSA UltraPure 1 mg ml 300 ul 1 25 ml ROX Reference Dye 100 ul 500 ul SYBR Green I 60 U ml Platinum Tag DNA polymerase 40 mM Tris HCl pH 8 4 100 mM KCL 6 mM MgCb 400 uM dGTP 400 uM dATP 400 uM dCTP 400 uM dUTP 40 U ml UDG and stabilizers Continued on next page Kit Contents and Storage continued Materials The following additional items are required for use with this kit Supplied by the User Coulter Counter or hemacytometer Microcentrifuge qPCR instrument Trypsin for adherent cell cultures only 1X cold phosphate buffered saline PBS without calcium or magnesium 0 2 ml thin walled PCR tubes or 96 well PCR plates Ice Pipettes Disposable gloves vi Introduction System Overview Advantages of the Kit The SuperScript III Platinum CellsDirect Two Step qRT PCR Kit with SYBR Green is an optimized kit f
16. me To prepare a master mix of ROX and Platinum SYBR Green qPCR SuperMix UDG 1 Add ROX Reference Dye to Platinum SYBR Green qPCR SuperMix UDG at a ratio of 1 ul of ROX for every 25 ul of SuperMix UDG Mix by vortexing for 10 seconds Store mixture at either 20 C or 4 C in the dark Use 26 ul of ROX SuperMix UDG mixture per 50 ul of reaction volume Note Use of ROX Reference Dye is not supported on the iCycler Rotor Gene Opticon and LightCycler platforms ROX Reference Dye is not required on the ABI PRISM 7900 BSA ultrapure non acetylated is included as a separate tube in each kit for use in LightCycler reactions qPCR Instruments Using PCR Tubes Plates Introduction This section provides a cycling program reaction mixture and protocol for qPCR instruments that use PCR tubes plates e g ABI PRISM Stratagene Mx4000 and Mx3000P Corbett Research Rotor Gene MJ Opticon For a protocol using the Roche LightCycler see page 13 Note This cycling program is recommended as a starting point and guideline Optimal cycling temperatures and times may vary for different target sequences primer sets and instruments After programming the instrument and preparing the reaction mix follow the protocol on the following page to perform the reaction gt ZN You can use 1 8 ul of cDNA template in the following protocol A Note depending on the concentration of the template Adjust the volu
17. me of water in the master mix accordingly for a final reaction volume of 50 ul We recommend 4 ul of template as a general starting point Cycling Program the qPCR instrument as follows Program 50 C for 2 minutes hold UDG incubation 95 C for 2 minutes hold 50 cycles of 95 C 15 seconds 60 C 30 seconds Melting Curve Analysis Refer to instrument documentation Master Mix Use the following table to prepare a master mix of all components except template and water Note Preparation of a master mix is crucial in qPCR to minimize pipetting errors Component 1rxn 50 rxns Platinum SYBR Green qPCR SuperMix UDG 25 ul 1250 pl ROX Reference Dye optional lul 50 ul Forward primer 10 uM 1u 50 ul Reverse primer 10 uM 1u 50 ul Autoclaved distilled water to 42 49 ul to 2100 2550 pl Final concentration 0 06 U l Platinum Tag DNA polymerase 20 mM Tris HCl pH 8 4 50 mM KCI 3 mM MgCh 200 uM dGTP 200 uM dATP 200 uM dCTP 200 uM dUTP 1 U UDG Volume of water used in the master mix will depend on template volume used in the reaction see steps 3 4 next page Continued on next page 11 qPCR Instruments Using PCR Tubes Plates continued Protocol 12 Program the qPCR instrument to perform a brief UDG incubation immediately followed by PCR amplification as shown on the previous page Optimal cycling temperatures and times may vary for different target sequences primer sets and instruments
18. n A Gerard G F 1998 Focus 20 30 17 Related Products Product CellsDirect Resuspension and Lysis Buffer E Gel Pre cast Agarose Gels 0 8 Starter Pak 1 2 Starter Pak 296 Starter Pak 496 Starter Pak UltraPure Agarose UltraPure Agarose 1000 100 bp DNA Ladder 123 bp DNA Ladder 1 Kb Plus DNA Ladder 18 Size 10 ml Resuspension Buffer and 1 ml Lysis Enhancer 9 gels and base 9 gels and base 9 gels and base 9 gels and base 100 g 500g 100g 50 ug 100 ug 250 ug 250 ug 1 000 ug Cat No 11739 010 G5000 08 G5000 01 G5000 02 G5000 04 15510 019 15510 027 10975 035 15628 019 15613 011 15613 029 10787 018 10787 026 Purchaser Notification Limited Use Label License No 1 Thermostable Polymerases Limited Use Label License No 5 Invitrogen Technology Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US 5 789 224 5 618 711 and 6 127 155 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim no right to perform any patented method and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed e
19. n the rotor of the LightCycler and run the program After cycling hold the reaction at 4 C until further analysis Collect and analyze the results Troubleshooting Problem Possible Cause Suggested Solution Cells in tissue culture wells do not detach burst Incubation temperature of lysis reaction is too low Incubate lysis reaction at room temperature instead of on ice No amplification curve appears on There is no PCR product Run the PCR product on a gel to determine whether PCR worked Then proceed to the troubleshooting the qPCR graph steps below No PCR productis Procedural error Confirm that all steps were followed Use the Control evident either in the RNA to verify the efficiency of the first strand qPCR graph or on a reaction see the next page on troubleshooting with gel the Control RNA RNA is degraded Add control total HeLa RNA to sample to determine if RNase is present in the first strand reaction A longer DNase I digestion can hydrolyze the RNA in the sample Use a digestion time of lt 10 minutes Maintain aseptic conditions to prevent RNase contamination Primer design is Verify your primer selection We recommend using suboptimal validated pre designed primers or designing primers using dedicated software programs or primer databases Target mRNA Maintain an elevated temperature after the annealing contains strong step transcriptional Increase the temperature of firs
20. ncerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 2010 Life Technologi
21. nd handling time e Total lysate volume is used in the cDNA synthesis reaction providing greater yields with a limited number of cells and allowing for detection of rare transcripts e SuperScript III Reverse Transcriptase with reduced RNase H activity and higher thermal stability produces high yields of cDNA in the first strand synthesis reaction for greater sensitivity and enhanced detection of rare transcripts e Platinum SYBR Green qPCR SuperMix UDG ensures optimal sensitivity and performance in qPCR using SYBR GreenI fluorescent dye with built in carryover contamination protection and a linear dose response over a wide range of target concentrations Continued on next page Introduction Continued Diagram of cDNA Synthesis from Cell Lysate SuperScript III RT Harvest cells Cell lysate First strand cDNA synthesis ORAL STTTT Treat with RNase H SCHE Single stranded cDNA MEL ue c e Proceed to real time quantitative PCR SuperScript III Reverse Transcriptase is an engineered version of M MLV RT with reduced RNase H activity and increased thermal stability The enzyme can be used to synthesize first strand cDNA at temperatures up to 55 C providing increased specificity higher yields of cDNA and more full length product than other reverse transcriptases Because SuperScript III RT is not inhibited significantly by ribosomal and transfer RNA it can effectively synthesize first str
22. nds 20 C second transition 92 C 0 seconds 0 1 C second transition continuous acquisition 40 C 0 seconds Use the following table to prepare a master mix of all components except template Note Preparation of a master mix is crucial in qPCR to minimize pipetting errors Component 1rxn 34 rxns Platinum SYBR Green qPCR SuperMix UDG 10 ul 340 ul BSA UltraPure 1 mg ml Tul 34 ul Forward primer 10 uM 1 ul 34 ul Reverse primer 10 uM 1 ul 34 ul Autoclaved distilled water to 18 ul to 612 ul Final concentration 0 06 U l Platinum Taq DNA polymerase 20 mM Tris HCl pH 8 4 50 mM KCI 3 mM MgCl 200 uM dGTP 200 uM dATP 200 uM dCTP 200 uM dUTP 1 U UDG Continued on next page 13 qPCR Roche LightCycler continued Protocol 14 Program the LightCycler to perform a brief UDG incubation immediately followed by PCR amplification as shown on the previous page Optimal cycling temperatures and times may vary for different target sequences and primer sets Set the fluorescence on the LightCycler to the F1 channel Prepare a master mix of all components except template as specified on the previous page For each reaction add 18 ul of the master mix to each capillary tube Add 2 ul of the cDNA from the first strand synthesis reaction Step 7 page 8 to each capillary tube for a final reaction volume of 20 ul and cap the tube Centrifuge tubes at 700 x 2 for 5 seconds Place reaction tubes i
23. nsferable license under U S Patent Nos 5 994 056 and 6 171 785 and under any corresponding foreign patent claims owned by Roche Molecular Systems Inc or F Hoffmann La Roche Ltd to use only this amount of the product for dye intercalation detection assays and related processes described in said patent solely for the purchaser s own internal research and development activities No license under these patents to use the PCR process is conveyed expressly or by implication to the purchaser by the purchase of this product Further information on purchasing licenses for these patented processes may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA No rights are conveyed expressly by implication or by estoppel under any apparatus or system claim of any patent or patent application ABI PRISM and GeneAmp are registered trademarks of Applera Corporation iCycler Mx4000 Rotor Gene DNA Engine Opticon and Smart Cycler are trademarks of their respective companies LightCycler is a registered trademark of Idaho Technologies Inc 21 Technical Service Web Resources Visit the Invitrogen web site at www invitrogen com for Technical resources including manuals vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invit
24. or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the most restrictive terms apply Life Technologies Corporation will not assert a claim against the buyer of infringement of patents that are owned or controlled by Life Technologies Corporation and or Molecular Probes Inc which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0354 Continued on next page Purchaser Notification continued Limited Use Label License No 276 Dye Intercalation Detection Assays Trademarks The purchase price of this product includes a limited non tra
25. or primer databases Primer contamination or truncated or degraded primers can also lead to artifacts Check the purity of your primers by gel electrophoresis If agarose gels are used we recommend cooling the gels before visualization with intercalating dyes Product detected at RNA is degraded Add control total HeLa RNA to sample to determine higher than if RNase is present in the first strand reaction expected cycle A longer DNase I digestion can hydrolyze the RNA number in the sample Use a digestion time of 10 minutes Maintain aseptic conditions to prevent RNase contamination Product detected at Template or PCR Isolate source of contamination and replace lower than expected carry over reagent s Use separate dedicated pipettors for cycle number contamination reaction assembly and post PCR analysis Assemble and or positive reactions except for target addition in a DNA free signal from no area Use aerosol resistant pipet tips or positive template controls displacement pipettors Unexpected bands Contamination by Do not omit the DNase Digestion step on page 7 For after gel analysis genomic DNA larger samples 71 000 cells use a longer DNase I incubation time i e up to 10 minutes Design primers that anneal to sequence in exons on both sides of an intron or exon exon boundary of the mRNA to allow differentiation between amplification of cDNA and products potential contaminating genomic DNA To test i
26. or synthesizing first strand cDNA directly from mammalian cell lysate without first isolating RNA and then amplifying the cDNA in a real time quantitative PCR qPCR reaction using Platinum SYBR Green qPCR SuperMix UDG In traditional qRT PCR RNA is first isolated from cells in a time consuming procedure that can lead to a loss of material Using the CellsDirect cDNA Synthesis System the cells are lysed and the cDNA is generated from the lysate in a single tube with minimal handling and no sample loss DNase I is added to eliminate genomic DNA prior to first strand synthesis After synthesis the first strand cDNA can be transferred directly to the qPCR reaction without intermediate organic extractions or ethanol precipitations This kit has been optimized for small cell samples ranging from 10 000 cells down to a single cell The use of SuperScript III Reverse Transcriptase ensures high specificity and high yields of cDNA from small amounts of starting material as little as 10 pg total RNA The use of Platinum SYBR Green qPCR SuperMix UDG ensures optimal qPCR performance using SYBR Green I dye with excellent sensitivity and a linear dose response over a wide range of target concentrations This kit has the following advantages e Compatible with a wide range of mammalian cell types grown under different treatment conditions e Cell lysis and first strand cDNA synthesis in the same tube minimizes reagent loss sample loss a
27. ou treat the cell lysate with DNase I to degrade any Digestion contaminating DNA 1 Place each tube plate from Step 15 page 5 or Step 7 page 6 on ice and add the following Component Amount DNase I Amplification Grade 1 U l 5 ul 10X DNase I Buffer 1 6 pl Mix by gently pipetting up and down or briefly vortexing and spin briefly to collect the contents Incubate the tube plate at 25 C or room temperature for 5 minutes Note A longer incubation time up to 10 minutes may be used for larger samples gt 5 000 cells However incubation times exceeding 10 minutes can greatly reduce cDNA yield Spin briefly and add 4 ul of 25 mM EDTA to each tube well on ice Mix by gently pipetting up and down and spin briefly to collect the contents Incubate at 70 C for 10 minutes Spin briefly and proceed to First Strand cDNA Synthesis page 8 First Strand cDNA Synthesis Required Materials First Strand cDNA Synthesis The following materials are provided by the user Thermal cycler preheated to 25 C Ice Pipettes The following materials are provided in the kit 2X RT Reaction Mix RT Enzyme Mix contains SuperScript III RT 100 units ul and RNaseOUT Recombinant Ribonuclease Inhibitor 20 units ul RNase H 2 U ul To each tube plate from DNase I Digestion Step 6 page 7 add the following Component Amount 2X RT Reaction Mix 20 pl RT Enzyme Mix 2 ul For negative RT controls use
28. rogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 E mail E mail 6117 tech_support invitrogen com jpinfoGinvitrogen com E mail eurotech invitrogen com SDS SDSs Safety Data Sheets are available on our web site at www invitrogen com sds Quality Control The Certificate of Analysis CofA provides detailed quality control information for each product The CofA is available on our website at www invitrogen com cofa and is searchable by product lot number which is printed on each box Continued on next page 22 Technical Service Continued Limited Warranty Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or co
29. t strand reaction up pauses to 55 C PCR product is qPCR instrument Confirm that you are using the correct instrument evident in the gel settings are settings dye selection reference dye filters but not on the qPCR incorrect acquisition points etc graph Problems with your See your instrument manual for tips and specific qPCR troubleshooting instrument Poor sensitivity Not enough Increase the number of cells used starting template RNA Higher than expected signal Too much first strand product was used in qPCR Decrease amount of the first strand product in qPCR Continued on next page 15 Troubleshooting continued Problem Possible Cause Suggested Solution Signals are present Template or Use melting curve analysis and or run the PCR in no template reagents are products on a 4 agarose gel after the reaction to controls and or contaminated by identify contaminants multiple peaksare nucleic acids To reduce the risk of contamination take standard present in the DNA cDNA precautions when preparing your PCR melting curve graph reactions Ideally amplification reactions should be assembled in a DNA free environment We recommend using aerosol resistant barrier tips Primer dimers or Use melting curve analysis to identify primer dimers other primer We recommend using validated pre designed primer artifacts are present sets or designing primers using dedicated software programs
30. ti Polymerase Antibodies Limited Use Label License No 223 Labeling and Detection Technology 20 Licensed to Life Technologies Corporation under U S Patent Nos 5 338 671 5 587 287 and foreign equivalents for use in research only The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity in a manner consistent with the accompanying product literature The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information
31. ulture vessel can be used e We recommend using a maximum of 10 000 cells per reaction Higher numbers of cells may inhibit reverse transcription and result in reduced yields and or truncated cDNA product Make sure that all solutions and equipment that come in contact with the cells are sterile Always use proper sterile technique and work in a laminar flow hood when handling cells The following materials are provided by the user e Mammalian cell cultures in growth media e Coulter Counter or hemacytometer e Centrifuge for pelleting cells e Incubator water bath or thermal cycler preheated to 75 C e Trypsin for adherent cell cultures only 1X cold phosphate buffered saline PBS without calcium or magnesium e 0 2 ml thin walled PCR tubes or 96 well PCR plates e Ice e Pipettes The following materials are provided in the kit e Resuspension Buffer e Lysis Enhancer DNase I Amplification Grade 1 U l e 10X DNase I Buffer e EDTA 25mM e Optional Control HeLa Total RNA All steps should be performed on ice and reagents should be chilled and or thawed immediately prior to use The incubator should be preheated to 75 C Continued on next page Lysing Cells continued Lysing Adherent Cells or Cells in Suspension Use the following lysis procedure for adherent cell cultures in vessels larger than 24 well plates For cells in suspension skip Steps 1 4 and proceed to Step 5 below 1
32. xpressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide
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