Hi all,
I am doing a few site-directed mutagenesis reactions and after digestion of the parent plasmid using Dpn1, I add the complete 50 uL reaction volume to 50 uL of NEB chemically competent cells for transformation. I follow the exact protocol for transformation as prescribed by NEB. Still, I see no colonies the next day. If follow the same protocol for plasmids, then I do get good colonies. I was wondering that in one case I have unmethylated nicked DNA and in the other case, I have double-stranded plasmid. Can that make a difference or do I have to vary the volume of the cells or the PCR reaction volume that I use for transformation?
Thanks in advance,
Prakriti
You should not add more than 10% of the cell volume of DNA to do the transformation. Do not add more than 5ul of your rxn to the cells.
Hanna Alalam Thanks for your suggestion. I will do a series of dilutions to see which condition works best for me.
You are better off just adding the max amount permissible which is 5ul for 50ul of competent cells. If that does not work then something is wrong with your reaction not the transformation.
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