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No PCR products?
i'm a student trying 2 get AGL20 gene products via PCR from the plant Piper sarmentosum Roxb (betel). I'm using AGL20 primers, 5'-ccc-cat-atg-gtg-agg-ggc-aaa-act-c-3' and 5'-ccc-gga-tcc-tca-ctt-tct-tga-aga-aca-agg-3'.
problem is however, that i have not been able to produce any pcr product bands even after >20 tries (absolutely no pcr products). the genomic dna samples used for pcr have been extracted using a modified CTAB method and show quite well on gel electrophoresis. i have tried variations with the PCR mix, including increasing or decreasing template/MgCl/dNTP/Taq/primer concentrations, but none give any results. hot starts, increasing denaturation and annealing times have also been tried. the annealing temperatures that i use range from 55-65 degrees C.
I know for a fact that the plant is capable of producing PCR products becos my senior has done it, although the products have all degraded.
so what r d problems that may be involved, n solutions?
You are using high GC content primers and your first primer has a major hairpin in it that likely is preventing it from hybridizing. Check it out... you have CCCC----GG-G-GGGG in that primer. You know the G's are going to bind to those C's in a hairpin. I think that first primer is your main concern, I really do. I'd redesign one that doesn't have that hairpin. Also with that GC content, you could try actually RAISING your annealing temp up to 70 or so.
Good luck, but I think you really do need to re-design that first primer.