sear in PCR no bands - (Aug/19/2006 )
Im trying to amplify 1.2kb fragment of phytoplasma(plant pathogen) DNA, in total plant DNA extrat using Phytoplasma specific primer set. But i only get a smear. it appears in the correct size on agarose gells. how should i improve the pcr to a band
thanks
chandima
please give us more details?
what is the concentration and purity of your template? have you spec'd it?
what are your primer characteristics?
have you tried any additives, such as DMSO, to improve specificity? what about using a gradient or touchdown PCR method?
what is the concentration and purity of your template? have you spec'd it?
what are your primer characteristics?
have you tried any additives, such as DMSO, to improve specificity? what about using a gradient or touchdown PCR method?
Im using the total DNA extract, ie plant dna+pathogen dna pcr used to work for several plant species at 200ng in 10 micro leter reaction but not for seceme the plant now im working with it gave faint band of correct size though (and no non-specific amplification) when i used only 50ng, could my dna impure????????
i have never tried any additives
Phytoplasma DNA is very AT rich. Try lowering the extension temperature from 72C to 66C and see if it helps. Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucleic Acids Res. 1996 Apr 15;24(8):1574-5. PMID: 862869