RT-PCR no amplification - (Jul/02/2008 )

I recently performed a PCR on some samples I already performed amplification
on, but htis time around none of the samples got amplified. I did not
forget to add any of the reagents, but the only difference in the way I
performed the experiment was I added the mastermixes to my plate and left
the plate uncovered for a very long time to get my samples prepared. I was
wondering if that could be a reason for no amplification. I also wanted to
know if repeated freezing and thawing of primers could affect the PCR
quality.

please help!!

there is no threshold value there and all of them come up N/A so assumed there is no product there not even my controls came up which is why i think either none of my samples ran anyways repeating it, hope sumtin comes up

QUOTE (gosala @ Jul 2 2008, 07:38 AM)

I recently performed a PCR on some samples I already performed amplification
on, but htis time around none of the samples got amplified. I did not
forget to add any of the reagents, but the only difference in the way I
performed the experiment was I added the mastermixes to my plate and left
the plate uncovered for a very long time to get my samples prepared. I was
wondering if that could be a reason for no amplification. I also wanted to
know if repeated freezing and thawing of primers could affect the PCR
quality.

please help!!

1. old, improperly stored, expired MM.

2. Machine breakdown; did your controls work?

3. Show your results run to an experienced colleague, it is possible that you may have data there, just can not see. No offense, this occurs with real-time machine/software too often

4. Primer F/T does not really matter, I have done it all the time, still, I prefer to keep primers at 4'C for a long time.