Wang et. al., Nature Biotechnology, April, 2000
Kate’s Protocol - Revised 4/23//02
Isolate Total RNA using Qiagen midi kit (Cat#75142) (see manufacturer's protocol) or by Trizol (Gibco BRL Cat# 15596-026) extraction (see manufacturer's protocol) or by PaxGene extraction (Pax tubes Cat# 762115, Pax Blood Extraction kit Cat# 762134) (see manufacturer's protocol). Resuspend total RNA in DEPC water at 1ug/ul concentration.
Speedvac RNA down to a total of 3-4ug in 9uL DEPC H20. Prepare RNA in PCR tubes and store at –80C overnight.
First strand cDNA synthesis:
In PCR reaction tube, mix
|
Ammt |
Reagent |
|
0.5-3ug |
total RNA |
|
9ul |
DEPC H2O |
|
1ul |
(1ug/ul) oligo dT(15)-T7 primer |
70C for 3min, snap cool on ice.
Add to PCR Tube (separately or in a Master Mix)
