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?
2007-02-15
01:01:09
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3 answers
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asked by
Anonymous
in
Biology