Bioscript Iii Reverse Transcriptase (Rtase)

BioScript III Reverse Transcriptase (RTase) is recombinant M-MLV RTase expressed in E. coli and purified to homogeneity. It has lower RNase H activity and high thermal stability. The enzyme is widely used to synthesize first-strand cDNA at temperatures up to 55A C with increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases. It can generate cDNA from 100 bp to 12 kb.
Component
BioScript III RTase(200 U/A A l)
Cat No. RE-12-001 10,000 U
5X RT Buffer 1000 A A
0.1M DTT 500 A A l
First-Strand cDNA Synthesis
1 In a sterile microfuge tube add:
RNA solution (10 pg~5 A g total RNA or 10 pg~500 ng mRNA)
1 Aul oligo(dT)20 (50 M), or other primers
1 Aul 10 mM dNTP Mix
nuclease-free H2O to final volume of 13 AuL.
2 Heat for 3-5 minutes at 65A C. Spin briefly and place promptly on ice.
3 Add:
4 A A 5X RT Buffer
1 A A 0.1M DTT
1 A A RNase Inhibitor (10 U/AuL,optional)
1 A A BioScript III RTase (200 units/A A l)
Final volume 20 AuL
If generating cDNA longer than 5 kb at temperatures above 50A C using a gene-specific primer or oligo(dT)20, the amount of BioScript III RTase may be raised to 400 U (2A A l) to increase yield.
4 Incubate at 50A C for 30AcA 60 minutes. Increase the reaction temperature to 55A C for gene-specific primer. Reaction temperature may also be increased to 55A C for difficult templates or templates with high secondary structure.
5 Inactivate enzyme at 70A C for 15 minutes.
6 Store products at -20A C or proceed to PCR using 2AuL first-strand cDNA synthesis reaction mix.
Amplification of some PCR targets (those >1 kb) may require the removal of RNA complementary to the cDNA. To remove RNA complementary to the cDNA, add 1 A A l (2 units) of E. coli RNase H and incubate at 37A C for 20 minutes.
Storage
Store all components at -20A C (non-frost-free). Thaw 5X RT BufferALA 0.1 M DTT at room temperature just prior to use and refreeze immediately.