Hello, I have DNA isolates which I used to performed PCR using MY011/09(~450bp outer) and gp5/6(~150bp nested) primers. The clean, specific PCR bands(~150bp) are then subjected to DNA purification with Zymo clean DNA recovery kit and sequenced with both gp5 and gp6 primers using ABI 7500. The purified PCR product is always run on gel electrophoresis, this is to check the quality and to ensure that DNA was not lost during purification.
I have tried several experiments but I could not get the sequences please advice further on what could be the problem. Thank you so much!
Kindest Regards,
Tawe
can we see a .abi file please? The sequencing may not have worked but sequence files contain much useful information about the run. What temperature (annealing) do you use for the pcr and also the sequencing. Is this a high GC template and what amount of amplimer did you use for sequencing and also what amount of each sequencing primer. Are these primers very old or quite recent?
Dear Saba,
there is a problem with your method in that both pcr primers are still present so the sequence will be a mixture of 2 sequences both forwards and reverse.
best wishes,Paul
I thought you were saying that after pcr there is no need to purify the product in which case both primers are still present in the reaction mix so just sequencing will produce a sequence from each primer so will look messy. Perhaps the company that did your sequencing purified your pcr product and then added just the one primer and then did the sequencing reaction. I am very happy that this worked successfully for you. best wishes
paul
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