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ISSR PCR issues

ISSR PCR issues - PCR - Polymerase Chain Reaction Forum

ISSR PCR issues - 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 10-02-2010, 08:50 PM
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Default ISSR PCR issues



Hello PCR community,
I知 a new grad student trying to amplify ISSR targets from an endangered fern in New York State. I知 using a master mix called AccrPrime from Sigma, 2% Formamide, 3Mm MgCl2 Primer pairs with Tm=44 and 20-50ng/ul of template. PCR conditions are 95C for 15 min, 94C for 1:30, 50C for 30sec, 72C for 1:30 for 40 cycles (lid is 95 at start). I知 trying to get as many bands as possible to compare genetic similarly between individuals. I致e check the product on 5%-7% acrylamide gels and 1%-2% Agarose (acrylamide stained with SyBr gold). I either get very faint bands or just smears no matter what conditions I optimize (Mg concentration, annealing temps, DNA concentration). I知 afraid this may be it unless I don稚 start producing some results. Some elements that may be an issue:
PCR kit (master mix, primers, reagent less buffers) are 兎xpired 5 months past
The primers, with such a low TM, may be too short and thus unstable
Acrylamide gels are not appropriate for DNA targets this size (1000bp is the clearest line)
I would really appreciate any suggestions as to what primers work well for ISSR amplification in ferns. Does it matter if they池e ferns (or just plants)? Where can I purchase them? Thanks you in advance.
Jeremy
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fern , issr , issues , markers , pcr , plants


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