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.
3. Add uL 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)
9. Transfer the column to a microcentrifuge tube. Apply 10uL of elution
solution, pre-heated at 75C, to the center of the filter. Wait 1min and
centrifuge for 30s.
10. Repeat with a second 10uL aliquot of pre-heated elution solution.