EXPRESS One-Step SuperScript
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1. 20 C for long term storage SuperMixes may be stored at 4 8 C for up to one month EXPRESS One Step SuperScript qRT PCR Universal 11781 200 11781 01K EXPRESS qPCR SuperMix Universal 5x5 ml ROX Reference Dye 500 ul 5 x 500 pl EXPRESS SuperScript Mix for One Step qPCR 5x1ml EXPRESS One Step SuperScript qRT PCR with 11791 200 11791 01K Premixed ROX EXPRESS qPCR SuperMix with Premixed ROX 5x5 ml EXPRESS SuperScript Mix for One Step qPCR iv Overview Introduction EXPRESS One Step SuperScript qRT PCR Kits provide components for one step reverse transcription and quantitative PCR qRT PCR in a convenient format that is compatible with both rapid and standard qPCR cycling conditions Both cDNA synthesis and PCR are performed in a single tube using gene specific primers and either total RNA or mRNA These one step qRT PCR kits have been formulated for use with fluorogenic probe based technology e g TaqMan probes or fluorogenic primers e g LUX Primers The RT mix includes SuperScript III Reverse Transcriptase and RNaseOUT Recombinant Ribonuclease Inhibitor in an optimized formulation All EXPRESS qPCR SuperMixes include Platinum Taq DNA polymerase MgCl dNTPs with dUTP instead of dTTP uracil DNA glycosylase UDG and stabilizers Note that this unique one step formulation includes a special heat labile form of UDG in the SuperMix to help prevent reamplification of carryover PCR products2. allowing cDNA synthesis from genuine target sequences ROX Reference Dye is either premixed in the SuperMix or included as a separate component to normalize the fluorescent signal between reactions for instruments that are compatible with this option ROX can adjust for non PCR related fluctuations in fluorescence between reactions and provides a stable baseline in multiplex reactions The following items are supplied by the user e Template RNA e Gene specific fluorescent primers or primer probe combinations e DEPC treated water e Microcentrifuge e Thermal cycler e Optional Normalization dye for instruments that do not use ROX e PCR tubes plates Instrument Compatibility Universal Kits Kits with Premixed ROX EXPRESS One Step SuperScript qRT PCR Universal includes ROX Reference Dye as a separate tube and can be used with a wide range of real time instruments including the following Applied Biosystems 7900HT 7300 7500 StepOne StepOnePlus GeneAmp 5700 and PRISM 7000 and 7700 Bio Rad MJ Research iCycler iQ i05 and MyiQ DNA Engine Opticon and Opticon 2 and Chromo4 Real Time Detector Cepheid Smart Cycler Corbett Research Rotor Gene 3000 Eppendorf Mastercycler ep realplex Roche LightCycler 480 Stratagene Mx3000P Mx3005P and Mx4000 EXPRESS One Step SuperScript qRT PCR with Premixed ROX can be used with real time instruments that are compatible w
3. ROX Reference Dye is supplied as a separate tube in the Universal Kits ROX is recommended for fluorescence normalization on Applied Biosystems instruments and is optional for Stratagene s Mx3000P Mx3005P and Mx4000 It is not required on other instruments ROX is composed of a glycine conjugate of 5 carboxy X rhodamine succinimidyl ester and is supplied at a concentration of 25 uM Use the following table to determine the amount of 25 uM ROX to use with a particular instrument Amount of Effective Fold Instrument ROX per 20 1 Concentration eee reaction of 25 uM ROX AB 7300 7900HT StepOne StepOnePlus 0 4 pl 50X 500 nM and PRISM 7000 and 7700 AB 7500 Stratagene Mx3000P Mx3005P and 0 04 pl 500X 50 nM Mx4000 Continued on next page Universal Kits Guidelines and Protocols continued Cycling Programs Universal Kits The following one step cycling programs have been developed as a general starting point when using EXPRESS One Step SuperScript qRT PCR Universal Program your real time instrument to perform cDNA synthesis at or above 50 C immediately followed by PCR amplification as shown below The fast cycling program is designed for the AB 7500 in Fast mode Note This mix is highly robust and can be used with a wide range of cycling programs on different instruments If you have an alternative program that you want to use you should test it wi
4. between reactions e SuperMix with Premixed ROX The qPCR SuperMix with premixed ROX includes ROX Reference Dye at a final concentration of 500 nM to normalize the fluorescent signal on instruments that are compatible with this option e Universal SuperMix The Universal SuperMix includes ROX as a separate component for instruments that use ROX at a different concentration or do not require ROX Continued on next page Overview continued Advantages of the Kits SuperScript lll Reverse Transcriptase Platinum Taq DNA Polymerase This highly robust one step formulation provides optimal convenience and sensitivity in qRT PCR with sensitive detection and a broad quantification range e SuperScript III Reverse Transcriptase has been engineered for reduced RNase H activity and increased thermal stability resulting in higher yields of CDNA e Platinum Taq DNA Polymerase provides an automatic hot start in PCR for increased sensitivity specificity and yield and has a short activation time for the rapid cycling of fast qPCR instruments e A special heat labile form of UDG in the SuperMix prevents amplification of carryover PCR products between one step reactions e The same SuperMix is also used in non one step EXPRESS qPCR kits available separately for maximum flexibility SuperScript III Reverse Transcriptase is a version of M MLV RT that has been engineered to reduce RNase H activity and provide increased
5. 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 21 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience New York Chou Q Russell M Birch D Raymond J and Bloch W 1992 Prevention of pre PCR mis priming and primer dimerization improves low copy number amplifications Nucl Acids Res 20 1717 1723 Kotewicz M L D Alessio J M Driftmier K M Blodgett K P and Gerard G F 1985 Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli Gene 35 249 258 Lindahl T Ljungquist S Siege
6. thermal stability for higher yields of cDNA Kotewicz et al 1985 The enzyme in this RT mix formulation can synthesize cDNA at a temperature range of 50 60 C Because SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA it can be used to synthesize cDNA from total RNA RNaseOUT Ribonuclease Inhibitor is included in the SuperScript mix to safeguard against degradation of target RNA due to ribonuclease contamination Platinum Taq DNA Polymerase is recombinant Tag DNA polymerase complexed with proprietary antibodies that block polymerase activity at ambient temperatures Chou et al 1992 Sharkey et al 1994 Activity is restored after the initial denaturation step in PCR cycling providing an automatic hot start in qPCR for increased sensitivity specificity and yield Continued on next page Overview continued Uracil DNA Glycosylase UDG ROX Reference Dye Additional Materials Required UDG and dUTP in the qPCR SuperMix prevent the reamplification of carryover PCR products between reactions Lindahl et al 1977 Longo et al 1990 dUTP ensures that any amplified DNA will contain uracil while UDG removes uracil residues from single or double stranded DNA The UDG used in the kit is a heat labile form of the enzyme that destroys any contaminating dU containing product from previous reactions prior to cDNA synthesis UDG is inactivated at temperatures of 50 C or higher thereby
7. 95 C refer to instrument manual for specific programming Continued on next page 13 Kits with Premixed ROX Guidelines and Protocols continued One Step qPCR Kits with Premixed ROX 14 Use the protocol below as a general starting point for one step qRT PCR Scale the reaction volume as needed for your real time instrument 1 Set up reactions on ice A standard 20 l reaction size is provided component volumes can be scaled as desired Always prepare a master mix of common components for multiple reactions 20 ul rxn EXPRESS qPCR SuperMix with Premixed ROX 10 pl Fluorescent primer probe mix conc and volume specified by manufacturer X ul EXPRESS SuperScript Mix for One Step qPCR 2 ul Template RNA e g 1 pg 1 ug total RNA 5 ul DEPC treated water to 20 pl 2 Prepare control reactions as follows No RT controls To test for genomic DNA contamination of the RNA sample do not add the EXPRESS SuperScript Mix No template controls To test for genomic DNA contamination of the enzyme primer mixes do not add template RNA 3 Cap or seal each PCR tube plate and gently mix Make sure that all components are at the bottom of the tube plate centrifuge briefly if needed 4 Place reactions in a real time instrument programmed as described on the previous page Collect data and analyze results 5 Optional The specificity of the PCR products can be checked by agarose gel electrophoresis Troubl
8. RESS SuperScript Mix for One Step qPCR 2 pl Template RNA e g 1 pg 1 ug total RNA 5 pl DEPC treated water to 20 pl See the table on page 9 for the amount concentration of ROX to use for your specific instrument 2 Prepare control reactions as follows No RT controls To test for genomic DNA contamination of the RNA sample do not add the EXPRESS SuperScript Mix No template controls To test for genomic DNA contamination of the enzyme primer mixes do not add template RNA 3 Cap or seal each PCR tube plate and gently mix Make sure that all components are at the bottom of the tube plate centrifuge briefly if needed 4 Place reactions in a real time instrument programmed as described on the previous page Collect data and analyze results 5 Optional The specificity of the PCR products can be checked by agarose gel electrophoresis 11 Kits with Premixed ROX Guidelines and Protocols Introduction This section provides a protocol and guidelines for one step qRT PCR using EXPRESS One Step SuperScript qRT PCR Universal Additional The following items are supplied by the user Materials e DEPC treated water Required Fluorescent primers probes see pages 6 5 for information Microcentrifuge Thermal cycler see page 4 for information on compatible thermal cyclers PCR tubes plates Premixed ROX ROX Reference Dye is included in the SuperMix at a final Concentration concentration of 500 nM whic
9. UV absorbance 2 Determine the OD260 of the solution using a spectrophotometer blanked against 10 mM Tris HCl pH 7 5 Calculate the amount of total RNA using the following formula Total RNA ug OD260 x 40 pg 1 ODs x 1 ml x dilution factor x total sample volume ml Example Total RNA was eluted in water in a total volume of 150 pl A 40 l aliquot of the eluate was diluted to 500 pl in 10 mM Tris HCl pH 7 5 An OD260 of 0 188 was obtained The amount of RNA in the sample is Total RNA ug 0 188 x 40 pg 1 OD260 x 1 ml x 12 5 x 0 15 14 1 ug Total RNA quality can be analyzed using a bioanalyzer such as the Agilent 2100 bioanalyzer with an RNA LabChip Alternatively total RNA can be analyzed by agarose gel electrophoresis RNA isolated using the PureLink kits or TRIzol Reagent typically has a 28S to 18S band ratio of gt 1 5 RNA is judged to be intact if discreet 28S and 18S ribosomal RNA bands are observed Universal Kits Guidelines and Protocols Introduction Additional Materials Required ROX Reference Dye Concentration This section provides a protocol and guidelines for one step qPCR using EXPRESS One Step SuperScript qRT PCR Universal The following items are supplied by the user e DEPC treated water e Fluorescent primers probes see pages 6 5 for information e Microcentrifuge e Thermal cycler see page 4 for information on compatible thermal cyclers e PCR tubes plates
10. cal 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 19 Purchaser Notification Limited Use Label License No 14 Direct Inhibition by Anti Polymerase Antibodies Limited Use Label License No 274 5 Nuclease Process 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 A license to perform the 5 nuclease process for research requires the use of a Licensed 5 Nuclease Kit containing Licensed Probe or the combination of an Authorized Core Kit plus Licensed Probe or license rights that may be purchased from Applied Biosystems This product is an Authorized Core Kit without Licensed Probe Its purchase price includes a limited non transferable immunity from suit under U S Patents and corresponding patent claims outside the United States owned by Roche Molecular Systems Inc or F Hoffmann La Roche Lt
11. ct to determine the optimal concentration in qRT PCR EXPRESS One Step SuperScript qRT PCR Kits were developed using TaqMan Gene Expression Assays provided at a 20X concentration as well as custom designed mixtures in which the final primer concentration was 500 nM each and the final probe concentration was 200 nM Continued on next page General Guidelines and Parameters continued Fluorescent LUX Primers available separately from Invitrogen are a Primers fluorescent primer based detection technology consisting of one gene specific primer labeled with a single fluorophore and a corresponding unlabeled primer The labeled primer is designed with the fluorophore near the 3 end in a hairpin structure that effectively quenches fluorescence prior to PCR making a separate quenching moiety unnecessary When the primer becomes incorporated into double stranded PCR product the fluorophore is de quenched resulting in a significant increase in fluorescent signal LUX Primers are available in pre designed formats www invitrogen com lux or can be designed for specific targets using the D LUX Designer www invitrogen com dluxdesigner Unlike fluorescent probe based technologies they are compatible with melting curve analysis A final concentration of 200 nM per primer is effective for most reactions Doubling the amount of reverse primer to 400 nM may improve the performance of certain reactions Optimal results may
12. d Roche for using only this amount of the product in the practice of the 5 nuclease process solely for the purchaser s own internal research and development activities This product is also an Authorized Core Kit for use with service sublicenses available from Applied Biosystems This product conveys no rights under U S Patents Nos 5 804 375 6 214 979 5 538 848 5 723 591 5 876 930 6 030 787 or 6 258 569 or corresponding patent claims outside the United States expressly by implication or by estoppel No right under any other patent claims such as apparatus or system claims and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is hereby granted expressly by implication or by estoppel This product is for research purposes only Diagnostic uses require a separate license from Roche Further information regarding the 5 nuclease licensing program may be obtained from the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Continued on next page Purchaser Notification continued Limited Use Label License No 5 Invitrogen Technology 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 academ
13. eshooting Problem Cause Solution No PCR product is RNA has been Confirm RNA degradation by evident in the qPCR damaged degraded bioanalyzer or running on a gel and graph or on a gel replace RNA if necessary RNase Maintain aseptic conditions contamination cDNA synthesis SuperScript II in this formulation temperature too typically operates ina temperature high low priming range of 50 C 60 C efficiency Primers are blocked Raise the incubation temperature by secondary and or redesign primers probes structure PCR product is qPCR instrument Confirm that you are using the evident on a gel but settings are correct instrument settings dye not in the qPCR incorrect selection reference dye filters and graph acquisition points Product detected at Inefficient cDNA Adjust cDNA synthesis temperature higher than synthesis and or primer design expected cycle RT inhibitors are Remove inhibitors in the RNA number present in RNA preparation by an additional 70 ethanol wash RNA has been Confirm RNA degradation by damaged degraded Bioanalyzer or running on a gel and replace if necessary RNase Maintain aseptic conditions contamination Inefficient PCR Optimize PCR conditions by amplification adjusting annealing temperature and or redesigning the primers Not enough Increase concentration of template template RNA RNA to 10 ng 1 ug total RNA Higher than Too much sample Decrease the concen
14. h is compatible with Applied Biosystems 7900HT 7300 StepOne StepOnePlus GeneAmp 5700 and PRISM 7000 and 7700 12 Continued on next page Kits with Premixed ROX Guidelines and Protocols continued Cycling The following one step cycling programs have been Programs developed as a general starting point when using EXPRESS Kits with One Step SuperScript qRT PCR Universal Program your real time instrument to perform cDNA synthesis at or above 50 C immediately followed by PCR amplification as shown below The fast cycling program is designed for the AB 7900HT and StepOne Note This mix is highly robust and can be used with a wide range of cycling programs on different instruments If you Premixed ROX have an alternative program that you want to use you should test it with this mix Note that your protocol must include an initial 250 C incubation step for UDG inactivation and cDNA synthesis Fast Cycling Program for AB 7900HT and StepOne 50 C for 15 minutes cDNA synthesis 95 C for 20 seconds 40 cycles of 95 C for 1 second 60 C for 20 seconds Optional Melting curve analysis fluorescent primers only 60 C 95 C refer to instrument manual for specific programming Standard Cycling Program 50 C for 15 minutes cDNA synthesis 95 C for 2 minutes 40 cycles of 95 C for 15 seconds 60 C for 1 minute Optional Melting curve analysis fluorescent primers only 60 C
15. hen working with RNA e Dedicate a separate set of pipettes buffers and enzymes for RNA work e Use RNase free microcentrifuge tubes If it is necessary to decontaminate untreated tubes soak the tubes overnight in a 0 01 v v aqueous solution of diethylpyrocarbonate DEPC rinse the tubes with sterile distilled water and autoclave the tubes TM You can use RNase Away Reagent a non toxic solution available from Invitrogen to remove RNase contamination from surfaces For further information on controlling RNase contamination see Ausubel et al 1994 Sambrook et al 1989 Continued on next page Template RNA continued Determining Total RNA Yield Determining Total RNA Quality Total RNA can be quantitated using the Quant iT RNA Assay Kit or UV absorbance at 260 nm Quant iT RNA Assay Kit The Quant iT RNA Assay Kit provides a rapid sensitive and specific method for RNA quantitation with minimal interference from DNA protein or other common contaminants that affect UV absorbance readings The kit contains a quantitation reagent and pre diluted standards for a standard curve The assay is performed in a microtiter plate and can be read using a standard fluorescent microplate reader UV Absorbance 1 Dilute an aliquot of the total RNA sample in 10 mM Tris HCl pH 7 5 Mix well Transfer to a cuvette 1 cm path length Note The RNA must be in a neutral pH buffer to accurately measure the
16. ic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product 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
17. invitrogen by technologies EXPRESS One Step SuperScript qRT PCR Kits For one step qRT PCR with SuperScript Reverse Transcriptase Catalog nos 11781 200 11781 01K 11791 200 and 11791 01K Rev Date 28 June 2010 Manual part no A10327 MAN0000689 ii Table of Contents Kit Contents and Storage c ccccccccceceseesesesnsnsesescseseecececeneteesesesnsnenenens iv OLE VICW sas niian aaia aA E E A a 1 Instrument Compatibility 00 0 ccc cece c cs eenensnssseeeesesensesesesesesesens 4 WAG OCS acesiessanntsavasicuactaneicccncnasuntecaivquandesangusececdaghincehied iccgdenaieeauete 5 General Guidelines and Parameters eseesssseeeceeeseeeceseseeeeneeaeeeees 5 Template RNA sssssccrssccssaticaesscoacdasiseeeagaanesenzsdsacscesageth EEEE SES EAAS Eria S ARESE 7 Universal Kits Guidelines and Protocols 0 0 0 eeseseeecrseeeeeneeseeeenes 9 Kits with Premixed ROX Guidelines and Protocols eee 12 Troubleshooting eene ea e eea aae R e ER e AER 15 Appendix cscs a eaceders tacasterececmwenndseentrenatanscenesacnava tanecuctedenaraveaianeundacs 17 Additional Products sx sauss esos paneer Sau eee dean bee ee 17 Technical Support genssa een tain a mera een eess 18 Purchaser Notification srece ne nE 20 REfELENICES niao a A A O 22 iii Kit Contents and Storage Kit EXPRESS One Step SuperScript qRT PCR Kits are shipped Components on dry ice The components in each kit are listed below and Storage Store all components at
18. ith ROX Reference Dye at a final concentration of 500 nM These include the following Applied Biosystems instruments 7900HT 7300 StepOne StepOnePlus GeneAmp 5700 PRISM 7000 and 7700 Methods General Guidelines and Parameters Reaction Setup and Conditions Fluorescent Probe Based Technologies e Starting material can be total RNA or mRNA e These kits use a two step cycling protocol with a denaturation step at 95 C and an annealing extension step at 60 C e Keep all components reaction mixes and samples on ice to prevent premature cDNA synthesis e Reaction volumes can be scaled from 5 pl to 100 ul depending on the instrument e For most templates efficient cDNA synthesis can be accomplished in a 5 minute incubation at 50 C For problematic templates or to increase the specificity of cDNA priming increase the cDNA synthesis temperature up to 60 C e For instrument specific guidelines see the section for each type of kit Fluorescent probe based technologies such as TaqMan Gene Expression Assays use two gene specific primers with a fluorescent labeled probe that is cleaved due to the 5 to 3 exo nuclease activity of Taq DNA polymerase thereby emitting a signal during PCR amplification These probe primer combinations may be packaged as predesigned gene specific assays or custom designed for a target of interest Consult the documentation provided with your fluorescent probe produ
19. nt Quant iT RNA Assay Kit o e RNA Quant iT RNA Assay Kit o e Kit 1 Oooo kit O Q83140 33140 TRIzol Reagent 100 Se 15596 026 200 ml 15596 018 PureLink 96 Total RNA Purification Kit 4 x 96 well plates 12173 011 17 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e 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 Invitrogen 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 website 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 6117 E mail E mail E mail tech_support invitrogen com _jpinfo invitrogen com eurotech invitrogen com SDS Certificate of Analysis 18 Safety Data Sheets SDSs are available on our website at www invitrogen com sds The Certificate of Analy
20. require a primer titration between 100 and 500 nM Melting Curve Melting curve analysis may be used with fluorescent Analysis primers to identify the presence of primer dimers and analyze the specificity of the reaction Note that melting curve analysis cannot be used with fluorescent probe based technologies Program your instrument for melting curve analysis using the instructions provided with your specific instrument Template RNA Input RNA General Handling of RNA Starting material can range from 1 pg to 1 ug of purified total RNA If you are starting with isolated mRNA the amount of template may be as low as 0 5 pg RNA should be free of RNase contamination and aseptic conditions should be maintained RNA may be treated with amplification grade DNase I see page 17 to remove any contaminating genomic DNA To isolate total RNA we recommend the PureLink Micro to Midi Total RNA Purification System TRIzol Reagent or the PureLink 96 Total RNA Purification Kit for high throughput applications see page 17 for ordering information When working with RNA e Use disposable individually wrapped sterile plasticware e Use aerosol resistant pipette tips for all procedures e Use only sterile new pipette tips and microcentrifuge tubes e Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin e Use proper microbiological aseptic technique w
21. rt W Nyberg B and Sperens B 1977 DNA N glycosidases properties of uracil DNA glycosidase from Escherichia coli J Biol Chem 252 3286 3294 Longo M Berninger M and Hartley J 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Ed Cold Spring Harbor Laboratory Press Plainview New York 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 2010 Life Technologies 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 22 invitrogen by Life technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
22. sis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Continued on next page Technical Support 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 concerns 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 typographi
23. th this mix Note that your protocol must include an initial 250 C incubation step for UDG inactivation and cDNA synthesis Fast Cycling Program for AB 7500 in Fast Mode 50 C for 15 minute hold CDNA synthesis 95 C for 20 second hold 40 cycles of 95 C for 3 seconds 60 C for 30 seconds Optional Melting curve analysis fluorescent primers only 60 C 95 C refer to instrument manual for specific programming Standard Cycling Program 50 C for 15 minute hold CDNA synthesis 95 C for 2 minute hold 40 cycles of 95 C for 15 seconds 60 C for 1 minute Optional Melting curve analysis fluorescent primers only 60 C 95 C refer to instrument manual for specific programming 10 Continued on next page Universal Kits Guidelines and Protocols continued One Step qPCR Universal Mix Use the protocol below as a general starting point Scale the reaction volume as needed for your real time instrument ROX is recommended for Applied Biosystems instruments and optional for Stratagene instruments see page 9 1 Set up reactions on ice A standard 20 l reaction size is provided component volumes can be scaled as desired Always prepare a master mix of common components for multiple reactions 20 ul rxn EXPRESS SuperScript qPCR SuperMix Universal 10 ul Fluorescent primer probe mix conc and volume specified by manufacturer Xul ROX Reference Dye 25 uM 0 4 pl 0 04 ul EXP
24. tration of expected signal added to reactions template RNA Continued on next page 15 Troubleshooting continued Problem Cause Solution Signals are present in no template controls and or multiple peaks are present in the melting curve graph Template or reagents are contaminated by nucleic acids DNA cDNA Use melting curve analysis fluorescent primers only and or run the PCR products on a gel after the reaction to identify contaminants See the guidelines for avoiding contamination on page 7 Primer dimers or other primer probe artifacts are present If you are using fluorescent primers use melting curve analysis to identify primer dimers melting curve analysis cannot be used with probes We recommend using validated pre designed primer probe sets or design them using dedicated software programs or primer databases Primer probe contamination or truncated or degraded oligos can lead to artifacts Check the purity by gel electrophoresis 16 Appendix Additional Products Additional Related products are available separately from Invitrogen Products Ordering information is provided below For more information visit our website at www invitrogen com or contact Technical Service page 17 TaqMan Gene Expression Assays visit www invitrogen com taqman RNase Away Reagent 250 ml 10328 011 DNase I Amplification Grade oon 015 Qua
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