I am amplifying target sequence 450 bp. I get single sharp band in the control and faint in sample with another sharp nonspecific product. why?
I need to get one single band from my sample to sequence the target . what should I change?
I changed annealing and DNA concentration, time of each cycle and used different PCR master mixes.
Since your pcr is working with the control it seems likely that the problem lies with the sample dna . I would test the od260/280 ratio and if this is lower than 1.8 you may need to re purify. It is also possible that the samples contain pcr inhibitors and sometimes using less dna works better as there is less inhibitor, You could try adding 1.5 times more magnesium than usual and run 1/2 1/4 1/8 dilutions of your dna and also add the normal amount of your sample dna to a positive control sample.If this dual dna sampe kills the pcr than you have an inhibitor problem and this will need to be dealt with
try to decrease cycles number beside that change annealing temp slightly below Tm of primers
make sure that your primers not self complement
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