Strange Maxi-Prep problem - low yields with QuickChange mutagenesis competent E. Coli (Aug/10/2006 )
Our lab has been running into problems when doing Maxi-Preps on the supercompetent E. Coli that come with the Quickchange mutagenesis kit.
Specifically, when doing side-by-side maxipreps with a wt version of a gene (grown up in our in-house competent cells) and a single bp mutated version of a gene (grown up in the kit cells). There was a 30-fold difference in yield between the two bact. types. I should point out that bacterial/nucleic acid pellets were the same size in both samples until the point in the protocol when NaCl/PEG was added to separate the plasmid DNA from the broken up genomic DNA, at which point the kit cells' pellet is much much smaller than its counterpart.
I did a second trial of side-by-side maxipreps, this time with our cells and the kit cells, both transformed with the mutant version of the aforementioned gene. Again, the pellet for the kit cells was much smaller than for our cells after the NaCl/PEG step, and there was a 12-fold difference in yield. We are only getting about 100 ug yield for the kit cells, so it's hardly worth it for a 300 mL culture and a fulls day's work.
I was just wondering if other people have run into this problem, or if anyone may know why this might be occurring. Oh yeah... the vector that the bacteria were transformed with carries ampicillin resistance, so the bacteria were grown in DYT containing amp.
Thanks for the help.
what are the genotypes of your cells and these of the kit?
do you use a dam- strin?
generally the dam- strin give less plasmid recovery than others. but don't know why...