I was performing a site directed mutagenesis of a plasmid, the goal was to change/insert 4 nucleotides into the original plasmid using primers as seen in the image:
Now I now there may be some deletions or nucleotides missing (one of the is expected anyways) but in the result and sequencing there are nucleotides missing from both the phosphorylated and the second primer, which I would not expect at all?
I used a 100 (nM) concentration of both primers in my reaction and phusion polymerase.
Any suggestions are appreciated. Thanks.
Is there a big difference in the Tm of the two primers? And have you adjusted the Tm according to the Phusion enzyme?
Are you adding enough dNTPs and magnesium? Low amounts could lead to mismatches.
Are the primers old \ have been frozen and thawed many times \ kept in water without any buffer for over a week? If so, your primers could be damaged.
Could your primers \ experiment be contaminated by exonucleases? The missing nucleotides might have been cut out.
I Hope I helped somewhat, best of luck!
The quality of the synthesis of the oligo is very important: maybe the company you ordered from is not very good: deletion inside synthetic oligo occurs when the protection reaction of the unreacted oligo is not 99.99%. we got this pb years ago; the company did not admit it was its fault... we change the supplier and get rid of the problem
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