We are doing site-saturation mutagenesis of LeuDH. But when we are mutating Q to G, the result of the sequence is always wrong, only a small fragment is the same to our templet plasmid. I have done it for three times, the results are the same. However, other mutation were all successful for the first time.
How are you doing your experiment? It is impossible to answer without knowing more details.
Michael J. Benedik I designed premiers and used PCR to invite the mutation. After the PCR, I verified the length of my plasmid using agarose gel electrophoresis and transformed my plasmid into Ecoli. I cultured Ecoli at 37℃. Then send my samples to the company for plasmid sequencing.
Btw, I had successfully mutated Q to G, but I tried more than 7 times only for this site mutation. Every time I used the same primers. The time I was about to redesign my primers, I succeeded.
So, is there any reasons to account for this? Or maybe just an coincidence?
It seems to me that you likely were not adequately removing the template DNA during the reaction, such as through DpnI treatment and hence you have a high background of the parent plasmid. The G3 sequence is just a poor quailtiy sequence so I would ignore that supposed mismatch, I doubt it is real.
Sometimes redesigning primers can help, especially if they are a bit longer, or sometimes one or the other primer just didn't synthesize well. So ordering new primers, especially given how inexpensive they are, is worth trying when the expt fails.
It is impossible to give you a definitive answer. It could be the primer, it could be the sequence around the region, it could be the mutation you are trying to make. But remaking the primer or adjusting its sequence slightly is the easiest thing to try.
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