Automated sequencing - Difficult gene (Mar/17/2009 )
Hello, I am trying to sequence a gene, however I can not amplify any exon by PCR. I have try everything, I have used betaine and it seems that it work, however, the Tm I am using to be able to amplify it are from 62 to 64 degrees, and after the sequencing reaction, there is any product. My target is quite rich in CGs and I believe that during the sequencing, which I run at 62 degrees (this is the ideal temperature for the sequencing enzyme). I believe that 62 degrees is not enough for my target to be amplified becaus of the CG content.
Does anybody has any suggestion how to sequence CGs rich targets? Can DMSO or betaine be used also in the sequencing reaction?
Thank you for your comments and best wishes.
we use betaine for our gc rich templates.
we anneal at 50C and extend at 60C as recommended by our kit (beckman-coulter genomelab dtcs quickstart).
the kit recommends 30 cycles but if the reaction is weak you can go up to 50 cycles before the taq has depleted too much.