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Re-using PCR plates?


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#1 Michelle0505

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Posted 23 June 2010 - 06:14 PM

Hi Everyone,

I just ran a qPCR experiment with the very last of my samples and am absolutely positive that I added all the reagents. The results, however, came out totally negative. I suspect that it was my enzyme, as it was sitting at room temperature for a while before I used it. If I can get new enzyme tomorrow, can I add it to the plate and re-run the experiment? I really needed this experiment to work, and I have no more cDNA or RNA to use...

Thanks!

#2 phage434

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Posted 23 June 2010 - 06:38 PM

PCR enzymes survive for hours at 95C. I sincerely doubt if a few hours at room temperature will do them any harm. The dNTPs, on the other hand, are probably heat sensitive. If you have used the last of your template DNA in these reactions, it would suggest to me that you are using too much DNA as template.

#3 Michelle0505

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Posted 24 June 2010 - 09:30 AM

I used the same amount of DNA template as I have used before, with successful experiments, I just meant that this was the last of my samples. If I added more dNTPs or more enzyme, or both, and reran the experiments, do you think it would work? In terms of the template DNA surviving a previous (unsuccessful) PCR cycle..




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