No specific was evident after amplification and primers didn't blast any other comparable sequence. This electropherogram was the same also after changing in PCR temperature (in order to improve specificity). Does anyone have a suggestion?
What do you exactly mean by "...three different electropherograms..."? Did you clone and sequence your amplification product and obtained completely different sequences or do you see double/triple peaks in your sequence chromatogram after direct sequencing of your PCR amplification product? If the latter is the case you might have identified a mutation in the gene.
I see a all sequence with a superimposed othe two different sequnces
In this case I would suggest that you clone your PCR product, e.g. using a TOPO TA or a pGEM®-T Cloning System, make plasmid mini preps of several colonies, sequence them and analyze the sequences.
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