2 replies to this topic

[Emma]

I'm having problems with TaqMan real-time PCR.

The system has worked for the past year but has suddenly stopped working. When I put on a PCR run, there is no amplification. I know that its not a problem with the PCR ready mix, cause I am able to amplify other genes with it. I also know that it is not a problem with my primers because I have carried out RT-PCR and there is amplification.

So, I think it may be a problem with the probe (possibly the fluorescent tag). I was just wondering if anyone has had similar problems or has any suggestion as to what is wrong? Also does anybody know of a way to test whether the fluorescent tag is working?

Thanks a mill!

Edited by Emma, 02 March 2005 - 07:00 AM.

[badcell]

A girl in my lab had the same problem a couple of years ago. All she was getting with her samples were erratic fluorescence readings well below the threshold level, just like the blank wells. It turned out to be that they had skipped a nucleotide during the synthesis of the probe, so it was not binding to the amplicons and she could not detect any amplification. So this things happen. Maybe you could just contact the company and ask if other users have the same problem (if it is a pre-designed probe).
Cheers!

[Emma]

Thanks for your reply!

I don't think this is the problem because the probe is not new. I have used it before and it worked well. But it sounds quite similar - the fluorescent readings were pretty much at zero, as if there was nothing in the wells. So I guess the problem must be with the probe. Its probably best to just order some more!