RT PCR avec SuperScript 3

Introduction : 

Description SuperScript™ ID Reverse Transcriptase is an engineered version of M-MLV RT with reduced RNase H activity and mcreased thermal stability.

The enzyme is purified to near homogeneity from E. coli containing the modified pol gene of Moloney Murine Leukemia Virus (1,2). The enzyme can be used to synthesize first-strand cDNA at temperatures up to 55°C, providing 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.

First-Strand cDNA Synthesis :

The following 20 µl reaction volume can be used for 10 pg-5 pg of total RNA or 10pg-500ng of mRNA.

1. Add the following components to a nuclease-free microcentrifuge tube :
- l µl of oligo(dT)20 (50 pM); or 200-500 ng of oligo(dT)12-18, or 50-250 ng of random primers; or 2 pmol of gene-specific primer.
- 10 pg-5 µg total RNA or 10 pg-500 ng mRNA
- l µl 10 mM dNTP Mix (10 mM each dATP, dGTP, dCTP and dTTP at neutral pH)
Stérile, distilled water to 13 µl

2. Heat mixture to 65°C for 5 minutes and incubate on ice for at least 1 minute

3. Collect the contents of the tube by brief centrifugation and add:
- 4 µL 5X First-Strand Buffer
- l µl 0.1 M DTT
- l µl RNaseOUT (Recombinant RNase Inhibitor. Note: When using less than 50 ng of starting RNA, the addition of RNaseOuT is essential.
- l µL of SuperScript 3 RT (200 units/µl) *If generating cDNA longer than 5 kb at temperatures above 50°C using a gene-specific primer or oligo(dT)20, the amount of SuperScript may be raised to 400 U (2 µl) to increase yield.

4. Mix by pipetting gently up and down. If using random primers, incubate tube at 25°C for 5 minutes.

5. Incubate at 50°C for 30-60 minutes. Increase the reaction temperature to 55°C for gene-spedfic primer. Reaction temperature may also be increased to 55°C for difficult templates or templates with high secondary structure.

6. Inactivate the reaction by heating at 70°C for 15 minutes.

The cDNA can now be used as a template for amplification in PCR. However, 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 l µl (2 units) of E.coli RNase H and incubate at 37°C for 20 minutes.