DNA amplification - troubleshooting - (Sep/24/2005 )

Hi everybody!

I am trying to amplify a small portion of mtDNA, using universal primers. During the optimisation process, I was able to put the system working. Two weeks later I have come back to the lab to finally start the reactions and from then on nothing worked. I have been doing all I can remember in terms of troubleshooting: using different reagents, different DNA concentrations (increasing and decreasing the amount of solution). Nothing. Thought on the possibility of having some salt left over from the salting-out method and did an ethanol re-precipitation and washing. Nothing. Any other suggestions?

Thanks a lot for your help!!!!

Tania

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Could the DNA have been degraded over the two weeks since it worked? You didn't say whether all attempts were made with the same aliquot of DNA template...

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I have thought about the option of degradation of the DNA, although I am not familiar with the reasons that it could have happened.

The first attempts that I made was with the same aliquots of DNA template. However, I did try with different ones as well. In both cases there was no amplification (with the samples 3 samples that had work before and with new ones - although coming from extravtions made on the same time).

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Well, you said you tried different reagents, so that likely rules out the enzyme. All that's static is the template and the primers. I assume your PCR machine is okay?

If a fresh extraction of template from the source doesn't do it, have the primers re-made, or redilute from a concentrated stock, if you have one.

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Thanks for the suggestions. The PCR machine is ok (been working on diferent things and no problem). The redilution of the oligos from the concentrated stock attempt was already made. I reckon it is definitly a problem with the extractions. The problem is I only had a few amount of tissue so the source is no longer there for new extractions! I guess I am stucked!!!

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