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fermentas RevertAid™ Premium First Strand cDNA Synthesis

说明
The RevertAid™ Premium First Strand cDNA Synthesis Kit is a complete system for highly efficient synthesis of first strand cDNA. The kit uses the RevertAid™ Premium Reverse Transcriptase (RT), an advanced enzyme derived by in vitro evolution of M-MuLV RT. The enzyme features the highest thermostability among the derivatives of M-MuLV RT and lacks the RNase H activity. The RevertAid™ Premium First Strand cDNA Synthesis Kit allows synthesis of long cDNA up to 20 kb at elevated temperature (up to 60°C) superceeding other systems in ability to prepare full length cDNA. Due to increased synthesis rates of the enzyme first strand cDNA synthesis, the reaction can be completed in 30 min.

RevertAid™ Premium Enzyme Mix contains RevertAid™ Premium Reverse Transcriptase and RiboLock™ RNase Inhibitor. RiboLock™ RNase Inhibitor effectively protects RNA templates from degradation by RNases A, B and C at temperatures up to 55°C.

Oligo(dT)18 and random hexamer primers are supplied with the kit. Random hexamer primers bind non-specifically and are used to synthesize cDNA from all RNAs in a total RNA population. The oligo(dT)18 primer selectively anneals to the 3’-end of poly(A) RNA, synthesizing cDNA only from poly(A) tailed mRNA. Gene-specific primers may also be used with the kit to prime synthesis from a specified sequence.

Water, nuclease-free is provided for reaction set-up and dilution of sample DNA. The absence of endo-, exodeoxyribonucleases, ribonucleases and phosphatases has been confirmed by appropriate quality tests.


质量控制
Functionally tested in RT-PCR using 100 fg of control GAPDH RNA and GAPDH control primers generate a prominent 496 bp product on 1% agarose gel after ethidium bromide staining.

组分
  • RevertAid™ Premium Enzyme Mix
  • 5X RT Buffer
  • dNTP Mix, 10 mM each
  • Oligo(dT)18 Primer
  • Random Hexamer Primer
  • Water, nuclease-free
  • Detailed protocol


Amplification of targets up to 20 kb in two step RT-PCR
图1。Amplification of targets up to 20 kb in two step RT-PCR.
1 µg of a total RNA from Jurkat cells (1 and 2) or a total RNA from mouse skeletal muscle (3 and 4) were used in a reverse transcription reaction with RevertAid™ Premium First Strand cDNA Synthesis Kit. Synthesized cDNA was used as a template in subsequent PCR with the Long PCR Enzyme Mix (#K0181) and primers specific for different genes:
3.9 kb – BCL2 (human B-cell CLL/lymphoma 2)
6.8 kb – POLE (human polymerase)
13.3 kb – Dmd (mouse dystrophin)
20.2 kb – Neb (mouse nebulin)
M – GeneRuler™ 1 kb Plus DNA Ladder (#SM1331)
Efficient cDNA synthesis at a wide temperature range
图2。Efficient cDNA synthesis at a wide temperature range.
1 µg of a total RNA from Jurkat cells were used in a reverse transcription reaction with RevertAid™ Premium First Strand cDNA Synthesis Kit using Oligo(dT)18 primer at different temperatures from 45°C to 60°C. Different length amplicons (0.5 kb, 3.9 kb, 6.8 kb and 9.4 kb) were successfully amplified using Long PCR Enzyme Mix (#K0181) from the cDNA synthesized at temperatures up to 60°C.
High synthesis rate at 50°C using RevertAid™ Premium First Strand cDNA Synthesis Kit
图3。High synthesis rate at 50°C using RevertAid™ Premium First Strand cDNA Synthesis Kit.
Synthesis of cDNA at 50°C for 5, 15, 30 and 60 minutes using 1µg of 7.5 kb RNA transcript as a template using RevertAid™ Premium First Strand cDNA Synthesis Kit and other kits for first strand cDNA synthesis. Reaction products were resolved on a 1% alkaline agarose gel. RevertAid™ Premium First Strand cDNA Synthesis Kit completed synthesis of 7.5 kb transcript in 5 min.
Higher sensitivity in two step RT-PCR using RevertAid™ Premium First Strand cDNA Synthesis Kit
图4。Higher sensitivity in two step RT-PCR using RevertAid™ Premium First Strand cDNA Synthesis Kit.
Total RNA from Jurkat cells (100 pg, 10 pg, 1 pg and 0.1pg) were used in reverse transcription reaction with RevertAid™ Premium First Strand cDNA Synthesis Kit at 50°C and other M-MuLV RT based kits at 42°C using Oligo(dT)18 primer in 20 µl reaction volume. 2 µl of RT reaction mixtures were used in PCR with Maxima™ Hot Start Taq DNA Polymerase (#EP0601) and primers specific for GAPDH gene.


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