qPCR troubles - (Nov/04/2008 )

Hi everyone,

I've got a very strange results for my last qPCR.
I had no determinated Ct value for any of the 6 cDNA samples X 7 cible genes X duplicat, and all the dissociation curves had several peaks.
Known that all the primers have been used for qPCR since 4 months and they worked perfectly. The SYBR mix was a new one just opend before use.

So now I am really confused. Is that because of cDNA's quality? Or the primers are degraded? Or something else?

Thanks

-yuchen-

Primers could be degraded, it looks like that. Try to prepare new stock or order a new ones and test them on this cDNA. And try some old cDNA you get gesults from before, on primers you use now. That should tell you where exactly is the problem.

-Trof-

QUOTE (Trof @ Nov 5 2008, 10:21 AM)

Thans for your reply.
I tried to migrate the cDNA on an agarose gel, and I clearly got a smear. I think that means the RT worked.
So now I am doing a PCR with the same primers on the cDNA I prepared and some old cDNA. If the PCR doesn't work for even the old cDNA then I can say the primers are probably degraded~~

-yuchen-

Hi! I'm new member here!

Please can you help me! I can't amplify my cDNA with stepone RT-PCR using SYBR green DyeI mix on plate with 96 wells. Ct not determined!
But, when I use conventional PCR, I amplify my cDNA!!!

Thanks

-ilham-

QUOTE (ilham @ Nov 19 2008, 06:41 PM)

I can't amplify my cDNA with stepone RT-PCR using SYBR green DyeI mix on plate with 96 wells.

What do you mean by stepone? are you using a kit or self-mix? Do you mean you are using a one-step realtime SYBR Green I mix kit? please clarify

-chrisbelle-

For each and every run (hell each and every scientific experiment) you need a positive and negative control.

Previous amplicons as a positive would at least show whether the PCR was working. Previous positive cDNA on top of that would show whether your new samples had any problems.

-maset-