I’m having a problem with a long range PCR. When I received my primers I tested them out, using a small volume of aliquotted and diluted primers, on a gradient to dial in Tm. They worked beautifully. After a couple days I then did my experiment (with a new aliquot of primers which I’ll call my working primers) and now no bands, just large dark streaks up near the wells (I’m assuming it’s due to secondary structure). I figure I made a mistake aliquotting my working primers so I re-ran the PCR, this time with the original test primers (as a positive control), plus my working primers, plus a newly made aliquot from the stock plates. This time the test primers (positive control) did not work as well as before. The working primers made another smudge, and my new aliquot from stock also made a smudge. What could have happened to these in 2 days time that they would work beautifully then not work at all?
Thanks for any suggestions,
This sounds like primer poisoning. What happens is you end up contaminating your work area with primer dimers and small fragments from your PCR and they kill the later PCR reactions. This is arely problem with long PCR. The solution is to make all new solutions and use different pippetors (go to a different lab to set up the reaction).
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