mutation during transformation? - (Aug/14/2006 )

Hi,

Is it possible that mutations are introduced into plasmids during transformation? I got two mutations and one deletion of my plasmid sequence after transformation. Before being cloned into the vector, the sequence of the PCR amplified fragments has been confirmed to be correct.

Thanks in advance for any help!

I have heard that mutations can occur during transformation but never experienced it ( i have made over 300 clones). And u say that there was 2 mutations and one deletion. thats too much. Something is wrong, for sure.

I would suggest sequencing other clones if u have.

Mutations can, and do, happen during transformation. The likelihood of mutations occuring seems to depend on the cloned sequence, where some GC-rich or hairpin sequences are more likely to have a base substitution, etc. In my experience, shRNA cloning was the only time I found a mutation in my insert, with 6% of clones having a mutation or incomplete sequence read (I was cloning many different shRNAs so the sample size was large ). The defect however could very well be in sequencing since this type of template is more difficult to analyze.

I have heard that with some plasmids you get recombination events when you grow the bacteria at 37°C. I was told that it depends on the plasmid. Therefore I grow some of my transformed ones at 30°C. Works well for me...