Description
    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 55℃ 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/μl) Cat No. RE-12-001  10,000 U
    5X RT Buffer                                                              1000 μl
First-Strand cDNA Synthesis
    1、In a sterile microfuge tube add:
          RNA solution (10 pg~5 μg total RNA or 10 pg~500 ng mRNA)
          1 µl oligo(dT)20 (50 μM), or other primers        
          1 µl 10 mM dNTP Mix                           
    nuclease-free H2O to final volume of 13 µL.
    2、Heat for 3-5 minutes at 65℃. Spin briefly and place promptly on ice.
    3、Add:
          4 μl 5X RT Buffer
          1 μl 0.1M DTT
          1 μl RNase Inhibitor (10 U/µL,optional)
          1 μl BioScript III RTase (200 units/μl)
           final volume 20 µL
    If generating cDNA longer than 5 kb at temperatures above 50℃ using a gene-specific primer or oligo(dT)20, the amount of BioScript III RTase may be raised to 400 U (2 μl) to increase yield.
    4、 Incubate at 50℃ for 30–60 minutes. Increase the reaction temperature to 55℃ for gene-specific primer. Reaction temperature may also be increased to 55℃ for difficult templates or templates with high secondary structure.
    5、 Inactivate enzyme at 70℃ for 15 minutes.
    6、 Store products at -20℃ or proceed to PCR using 2µL 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 μl (2 units) of E. coli RNase H and incubate at 37℃ for 20 minutes.
Storage
    Store all components at -20℃ (non-frost-free). Thaw 5X RT Buffer、0.1 M DTT at room temperature just prior to use and refreeze immediately.