cDNA
preparation
(Promega)
(V. Orgogozo, 2007)
1. Prepare tubes containing 14.5uL of RNA isolated with the SV RNA
Isolation System, Promega (total 2-5ug of RNA) + 0.5uL oligo-dT18
(30pmol/uL).
2. Incubate at 70C in the PCR machine for 5min and then place
immediately on ice (RTE programm on the PCR machine).
3. Add 10.5uL of the mix in each tube.
mix = 5uL 5x RT buffer
1.25uL
dNTP (10mM)
0.75uL
Rnasine
1uL
Reverse-Trancripase M-MLV (200u/uL)
2.5uL
DEPC water
4. Incubate at 42C for 60min.
5. Denaturate DNA-RNA hybrids at 94C for 5 min.
9. Put on ice immediately and store at -20C. Use 1uL of cDNA for a
25uL-PCR reaction.