Hello researchers,

I am currently trying to generate a bank of mutants of a yeast gene I am working on. After performing a error-prone PCR based on dNTPs imbalance, I clone the mutated fragments in a pBAD43 plasmid. I then use the EcoRI-HF and HindIII-HF to restrict fragments and plasmid, and T4 for ligation, performed O/N at 16°C. I generally use 5µL of ligation mix (out of 20) to transform my E. coli by electroporation (I should mention that I don't use ultracompetent cells, because I need a specific coli strain for a later screening method).

With this strategy, the number of colonies I get is quite low... Using 40ng of open plasmid in my ligation mix, I get approx. 200 colonies (whatever the insert:plasmid ratio), and 500 with 80ng. Generating a bank of mutants would require hundred of thousand of clones though, meaning I need to drastically improve my cloning efficiency!

Do you have any suggestions about this cloning strategy? I'm thinking about increasing the amount of open plasmid in ligation mix (to 200ng?), inactivating the T4 ligase before transformation, using more than 5µL of ligation mix to transform bacteria.

Or maybe should I use another cloning method? Some people in my lab use USER or Gateway cloning but I never did myself, so I am not really aware of their efficiency...

Thanks a lot for reading/helping ! :)