Protocol The following protocol is provided for rst strand cDNA synthesis using RevertAid H Minus Reverse Transcriptase
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Protocol The following protocol is provided for rst strand cDNA synthesis using RevertAid H Minus Reverse Transcriptase

All reverse transcriptases RT are suitable for the synthesis of fulllength 57375rst strand cDNA but they differ in reaction temperatures amounts of RNA transcribed sensitivity and RNase H activity The table below indicates reaction conditions recomm

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Protocol The following protocol is provided for rst strand cDNA synthesis using RevertAid H Minus Reverse Transcriptase




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Protocol The following protocol is provided for rst strand cDNA synthesis using RevertAidô H Minus Reverse Transcriptase. All reverse transcriptases (RT) are suitable for the synthesis of full-length rst strand cDNA, but they differ in reaction temperatures, amounts of RNA transcribed, sensitivity and RNase H activity. The table below indicates reaction conditions recommended for each RT. Enzyme units and RNA amounts are provided for 20 l of RT reaction volume: Reverse transcriptase Reaction temp. Active up to Reading length RHase H activity

Inactivation Units Total RNA poly(A) RNA Maxima RT 50-55C 60C 20kb 85C, 5min 200 10pg-5g 0.1pg-500ng RevertAid 50-55C 60C 20kb 85C, 5min 200 10pg-5g 1pg-500ng RevertAidô H 42-45C 55C 13 kb 70C, 10min 200 0.1ng-5g 10pg-500ng RevertAidô RT 42C 50C 13kb 70C, 10min 200 0.1ng-5g 10pg-500ng M-MuLV RT 37C 37C 9kb 70C, 10min 40 100ng-5g 10-500ng AMV RT 45-60C 60C 13kb ++ 85C, 5min 10 10ng-5g 1-100ng

Master Mix. To prepare several parallel reactions and to minimize the possibility of pipetting errors and contami nation, prepare a RT master mix by adding all reaction components except RNA into one vial. Prepare enough master mix for the number of reactions and add one extra to compensate for pipetting errors. Sample template RNA into individual tubes and keep on ice. Aliquot the prepared master mix into tubes with RNA. Mix and briey centrifuge all components after thawing, keep on ice. 1. Add into sterile, nuclease-free tube on ice in the order given: poly(A)+ mRNA or 1 g

Total RNA or 5 g Specic RNA transcript 0.5-1 g Oligo(dT)18 primer (0.5 g/l) or 0.5 g Random hexamer primer (0.2 g/l) or 0.2 g Gene-specic primer 100 pmol DEPC-treated Water to 11.5 l Total volume 11.5 l 2. Optional (if RNA template is GC-rich or is known to contain secondary structures). Mix gently and briey centrifuge, incubate at 65C for 5 min, chill on ice and briey centrifuge, then place the tube on ice. 3. Add in the order given: 5X reaction buffer for reverse

transcriptase 4 l RiboLockô RNase Inhibitor 0.5 l (20 u) dNTP Mix, 10 mM each 2 l (1 mM nal concentration) RevertAidô H Minus Reverse Transcriptase 1 l (200 u) Total volume 20 l This protocol is for the First Strand cDNA Synthesis
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thermofisher.com thermoscientific.com  2012 Thermo Fisher Scientic Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientic Inc. and its subsidiaries. Specications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Protocol 4. Mix gently and briey centrifuge. 5. If oligo(dT)18 primer or gene-specic primer is used, incubate 60 min at 42C. If random hexamer primer is used, incubate 10 min at 25C followed by

60 min at 42C. For reverse transcription of GC-rich RNA reaction temperature can be increased up to 45C. 6. Terminate the reaction by heating at 70C for 10 min. The reverse transcription reaction product can be directly used in second strand cDNA synthesis or stored at -20C.