Berglund:RT-PCR

RT-PCR is the method of reverse transcribing RNA to single stranded DNA and from single stranded DNA into double stranded DNA.

We have two common methods that we use that have worked well for us in generating product from mRNA, transcribed RNA, spliced RNA, in vivo work, etc.

The methods here are shown from SELEX RNA's and the primer concentrations have been optimized. Optimization of primer concentrations is critical for RT to work.

Reverse transcribing.
AMV-
6µl 5xAMV buffer
15µl 20µM N30.2-R
6µl 10mM dNTP
7.4µl RNA

Heat to 100C for 2 min
add 0.9µl SS to the experimental
incubate at 42C for 1 hr

Superscript II (Gibco)

5µl SSII Buffer
15µl 20µM R Primer
5µl 10mM dNTP
3.4µl 0.1M DTT
10µl RNA

Heat to 100C for 2 min
add 0.9µl SS to the experimental
incubate at 42C for 1 hr

PCR
71.5µl water
13.3µl 10xPCR buffer
3.3µl 20µM N30.2-F
2.6µl 10mM dNTP
2.6µl Lab Taq
Mix on ice, preheat the thermocycler, place tubes into hot thermocycler for "hot start".

PCR program
95C for 2 min
95C for 45 sec, 57C for 45 sec, 72C for 1 min
72C for 5 min
4C for infinity

Check on gel (3% agarose for products >100, 2% if >300, 1% for everything else.)