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Problematic sample...

Problematic sample... - PCR - Polymerase Chain Reaction Forum

Problematic sample... - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 03-06-2012, 05:54 AM
Pipette Filler
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Default Problematic sample...



Greetings all-

We have a rare sample that was obtained from a museum skin scraping that we have been trying to amplify for five different genes. The only success we've had is an, ever-so-slightest, single band from RAG1 Section 1 (not sequenced yet, but we're not holding out any hope...it's that faint). The rest of the PCR reactions with this sample have failed miserably. This sample is part of a very large project and we're not new to these genes or this group by any means.

While trying to figure out what is going on with this sample, which has now been extracted on three different occasions, we noticed a somewhat odd pattern today. On every gel image that this sample has been on, all most all samples have worked perfectly and have since been sequenced with no problems. However, on all these gel images their is clear evidence of primer dimer formation/clouding at the end of each lane. Again, not a problem, as the products have always sequenced cleanly etc. However, we noticed today that on each of these PCR runs with this problematic sample, the lane in which this sample was ran has not only not yielded any band, but there is also no primer dimer formation/cloud in this lane. We're talking at least 10 reactions here. We have had other failures on some of these PCRs, but in each of those cases, the primer dimer/cloud were always clearly present at the appropriate length. But not this sample which is just a solid black lane.

Anyone have any clues as to what might be causing this? We were thinking that maybe there is some chemical in the skin (from when it was preserved) that might be somehow blocking the function of the DNA polymerase every time. We also suspected that maybe there was some kind of phenol carry over from the extraction process, but at this point it has been extracted three times, each time with extreme care due to it's importance.

Any suggestions would be great. At this point, it's more of a nagging curiosity than anything.

Thanks!
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Old 03-09-2012, 11:33 PM
Pipette Filler
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Default Re: Problematic sample...

Have you tried purifiying the sample after the extraction (before the PCR)? The absence of primer dimer somethimes also happens when there is amplification, althought non detectable through agarose electrophoresis. You could try doing your PCR the way you normaly do and then take a small amount (1ul...or you could even do a input gradient) of this "PCR product" and use it as input for a second PCR. You can use the same protocol for this secondary PCR, just lower the cycle number. Good luck!
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