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.)