Hi, I'm a relative PCR newbie and I have been learning and honing the process. Now I am trying some novel sequencing but I seem to be getting some issues with PCR of NADH from insect mtDNA. I use extraction and PCR cycle protocols from the literature and have honed the template concentration using runs with different dilutions.
However, my gel and sanger sequencing results are strange. I get bright bands of the relative right size (roughly 550 bp and 330 bp). However these bright and tight bands gradual diffuse and fade as the gel runs while my ladder does not. An attempt and sequencing and BLAST shows a match for the desired sequence and organism I am looking at but at a much shorter length (100 bp) with the rest being garbage. I have run into this situation using PCR protocols from the literature and touchdown PCR.
Can you attach an original sequencing file please. It Is possible that your short sequence is simply that in each direction you cannot sequence the primer or the next 40 bases with any accuracy. One reason for short sequences is too much template in the sequencing reaction so the reagents get used up mainly at the start of the sequence. Your bands are diffuse so are either being run much too fast ( lower the running voltage) or are very non specific. Touchdown may not be needed and a higher annealing temperature might help this. Possibly also you are not giving the gel time to cool properly so the edge where the ladder is has set but the middle is not fully set.
Can you attach an original sequencing file please. It Is possible that your short sequence is simply that in each direction you cannot sequence the primer or the next 40 bases with any accuracy. One reason for short sequences is too much template in the sequencing reaction so the reagents get used up mainly at the start of the sequence. Your bands are diffuse so are either being run much too fast ( lower the running voltage) or are very non specific. Touchdown may not be needed and a higher annealing temperature might help this. Possibly also you are not giving the gel time to cool properly so the edge where the ladder is has set but the middle is not fully set.
I appreciate the advice Paul. Let me try gradually raising the temp and lowering the template concentration.
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