Hi all. I seem to have a serious problem with primer dimer formation. I am not sure but I believe it is for this reason that my PCRs aren't working lately. Is there anything I can do to completely get rid of them while not compromizing the product? I need my products so bad but the bands are either faint or absent from my agarose gels. please help
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Hi, if you can't redesign your primers, try to optimize your PCR:
if your primer stock has gone through a lot of freeze-thaw cycles, heating them at 99 degC for 5 min, followed by shock chilling them in ice water or liquid nitrogen might help, too. HTH Wo |
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I have recently heard of a similar situation that was corrected by merely ordering new primers after it was found that too much freeze-thawing of primers was the problem. It appears that you are saying that your PCR was working at one point, then stopped recently. Sounds like time to re-order primers (luckily this may be a cheap fix). JMG |
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If you have faint bands, have you tried reamplifying using the band as the template? |
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Check the pH of your buffers/water (yes, even the water). In my experience, a lot of annoying problems can be resolved by correcting the pH of the reaction. |
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