Has anyone out there tried out NEB's SDM kit? I am using it to make a couple of deletion and substitution mutants but I seem to be getting only wild type colonies and none with my constructs of interest, despite designing primers with their online tool and following the manufacturer's instructions to the letter!! Can anyone help me out? Any suggestions?
I had not try NEB site directed mutagenesis kit. But I did tried using QuickChange Lightning SDM kit. But, I prefer using overlapping PCR to perform SDM. I used NEB Q5 high fidelity DNA polymerase and using primers containing desired mutations (length roughly 30 - 35 b). Most of the clones I got consists of the desired mutation. Perhaps you can try using this method. The only disadvantages of this method is you need to clone again. For me, I rather do more hard work than getting no desired clone. Good Luck.
You may have too much of the original plasmid in the mix. You may also obtain better results with prolonged incubation time (several hours) in the phosphorylation, ligation, Dpn cleavage step.
IF the O/N KLD incubation didn't work well, try to modify the ratio of primer: template used in your first PCR.
Changing this ratio to favour template-primer interaction will reduce considerabily your wild type colonies.
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