I maked some PCR of rpoB gene (Mycobacterium tuberculosis). I obtained my interest fragment of 411 bp together with some unspecifics bands that disturb sequencing step: the qualities of chromatograms are bad. Before I perform purification, I want to know if the dilution of a such PCR product can improve sequencing.
normally I would always do gel extraction for gettting a nice and clean sequencng template. By doning this you know the size and that you sequence the right fragment
I suggest you to change a Magnesium concentration for exemple. This can increase the specifity of the reaction. Excess magnesium results in accumulation of nonspecific amplification productcs seen as multiple bands. Or you also can made a gel extraction.
Hi Amel,
You can do a gel extraction or also 'Band Stab PCR'....see the Original refernce...it is a useful technique to re-amplify a PCR band from a set of multiple bands
regards
Try to perform PCR by increasing the annealing temperature by 2. This will help you in getting a single band. Other wise run the gel for a longer time so that the unspecified bands seperate out and then cut the required fragment and proceed with gel elution and sequencing.
It is helpful of increasing the annealing temperature by 2, cut right size band from gel and PCR again also work.
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