No colonies mean all your methylated parental strands are being eliminated. I think your plasmid concentration need to be increased for higher efficiency.

And, if you have NEB's Q5 pol and Dpn1 available, you can try SDM manually.

Hi, Papri,

Aren't the methylated parental plasmids meant to be eliminated by MrcBC endonuclease (already present in DH5a cells) in the first place. Anyways the methylated parental plasmid was not mutated, the daughter plasmids were. What about those daughter plasmids which were mutated, why didn't they transform.

Moreover, the recommended plasmid concentration to be taken for mutagenesis PCR was 25ng/ml, I already increased it to 50ng/ml.

Thanks for the reply,

Regards

Dear Diptesh,

I've performed many SDMs manually, so I can share my experiences regarding that only. Almost every time, I got only 2 out of 10 colonies positive after sequencing, so 80% were false colonies. And, I use 50ng in total 100ul of PCR reaction manually.

Although the kit you are using is really very efficient, you might need to standardise your protocol first.

Best regards.

Papri

The parental plasmids need to be digested by Dpn1 after SDM reaction, and then transformed into DH5a competent cells (lab prepared).