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problem with site directed mutagenesis
Posted by: randa32 (IP Hidden, New member, 1)
Date: February 11, 2008 12:30PM
Hello,
I'm performing site directed mutagenesis of a single amino acid on extremly GC rich DNA. I'm using the quick change protocol with some changes to the PCR cycles and I have had success in the past. After the PCR, there is a Dpn1 digestion followed by XL1blue transformation. I do get colonies and grow to make a miniprep. I did a digestion followed by an agarose gel and the bands were a little off but I was still seeing two bands so I sent the samples for sequencing. But the sequencing only shows the vector. I don't know what happened to my parent DNA. I've tried altering the template and primer concentrations. I'm not sure which step is the problem since it is hard to check at each point. Any suggestions would be great, I think my primers are ok but I'm going to try designing new ones too. Thanks!
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