First-Strand cDNA Synthesis
1. In a sterile microfuge tube, first 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°C. Spin briefly and place promptly on ice.
- 4 μl 5X RT Buffer
- 1 μl 0.1 M DTT
- 1 μl RNase Inhibitor (10 U/μl)
- 1 μl GScript RTase (200 units/μl)
- final volume 20 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 GScript RTase may be raised to 400 U (2 μl) to increase yield.
4. Incubate at 50°C for 30-60 minutes. Increase the reaction temperature to 55°C for gene-specific primer.
The reaction temperature may also be increased to 55°C for difficult templates or templates with high secondary structure.
5. Inactivate enzyme at 70°C for 15 minutes.
6. Store products at -20°C or proceed to PCR using 2 μl first-strand cDNA synthesis reaction mixture. Amplification of some PCR targets (> 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°C for 20 minutes.
Store all components at -20°C (non-frost-free). Thaw 5X RT Buffer, 0.1 M DTT at room temperature just prior to use and refreeze immediately.
|MSDS||GScript RTase||0.1M DTT||5X RT Buffer|