Problems for saturation mutagenesis library - (Jul/28/2011 )
phage434 on Sat Jul 30 14:02:54 2011 said:
Yes, and the best would be a T7 promoter which will not be active in the cloning strain at all. What is the native AA in that position?
Alanine
-Biogareth-
So, perhaps 50% of your mutants still have an alanine in that position; a large fraction of the remainder have a glycine. Are you sure you are removing unmodified template from your reactions? Carryover of the unmutated DNA would explain a lot of what you are seeing. If this is being done with PCR, then perhaps you are using too much template. You could also perhaps remove template by DpnI digestion following PCR amplification and prior to cloning.
-phage434-
phage434 on Sat Jul 30 23:19:04 2011 said:
So, perhaps 50% of your mutants still have an alanine in that position; a large fraction of the remainder have a glycine. Are you sure you are removing unmodified template from your reactions? Carryover of the unmutated DNA would explain a lot of what you are seeing. If this is being done with PCR, then perhaps you are using too much template. You could also perhaps remove template by DpnI digestion following PCR amplification and prior to cloning.
Yes, DpnI digestion step is always performed after PCR amplificaiton. Most of unmodified template is supposed to be removed by this step. I don't think it contains quite a lot of unmutated DNA, otherwise the other two bases should be dominately by wild-type bases (but in my case, both positions are equally distributed).
-Biogareth-