Nonspecific PCR amplification? - (Apr/11/2006 )
As a new role in PCR domain,I recently encount some problems.
Please view my attached file.
The parameters as follow:
PCR-SSP for single nucleotide polymorphisms(SNP):
Pri for specific:1.0uL(0.4uM)
35 cycles in all:
The Tm of the Primers(8 pairs):
As showed in the graph:
lane 1-2 for system 1;
lane 3-4 for system 2;
lane 5-6 for system 3;
lane 7-8 for system 4.
All but lane 8 are bad enough,could the problem of the results for other lanes (1-7) are caused by nonspecific?
Thank you very much in advance for your careful reading and suggestion.
It looks lifrom the gel like there is too much template in there. I'd try with a tenth of what you put in
Also make sure you have twice as much of the common primer compared to the (presumaby) allele specific primers, eg 1ul common primer, 0.5ul each of allele specific primers
you might also want to reduce the temperature of your annealing step. normally it should be lower than the lower tm of your primers.