ISSR PCR issues

[jjdiscen]

Hello PCR community,
I’m a new grad student trying to amplify ISSR targets from an endangered fern in New York State. I’m 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’m trying to get as many bands as possible to compare genetic similarly between individuals. I’ve 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’m afraid this may be it unless I don’t start producing some results. Some elements that may be an issue:
• PCR kit (master mix, primers, reagent less buffers) are “expired” 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’re ferns (or just plants)? Where can I purchase them? Thanks you in advance.
Jeremy