I'm doing RT PCR, and i'm stuck.
here's the thing... i've got two pairs of primers, one pair for my gene and one pair for my control (cyclophylin).
I took 1ug of RNA, made cDNA, and ran a normal PCR (Taq) with these primers. I got the right size bands.
Our lab is currently inbetween funding at the moment, and although it's been recommended that we purchase a sybr green premix kit, we can't afford to at the moment. but we do have a premix, just without the sybr green... so i mixed in some sybr green into his premix, according to protocol.
i ran a test real time RT PCR just to get the standards, and to see if anything needed tweeking.
it needs tweeking.
first time, no product showed up whatsoever. melting curve all over the place.
second time, no product, but an ok melting curve.
third time, no product, but an ok melting curve.
I decided to test out the reaction on a gel, see whatsup. Using the same ingredients as the RT PCR.
There is no product. (swear kick swear kick).
What should I do now?
I can't think of what to do next.
Edited by bioforum, 02 February 2005 - 08:02 PM.