competent cells question - (Jun/20/2005 )
does anybody here have experience with different competent cells? Right now I am using DH5alpha's, but I'm having a problem with them.
I can perfectly grow a plasmid in them with repeats (talking about pNL4.3, a molecular clone of HIV which has two retroviral LTR's in it).
However, when I transform a ligation, I always get very bizarre results. After restriction analysis I never get the right fragments, and the fragments I get can not be explained by self-ligation either.
Here's the strategy I use:
I do cut my vector with EcoRI and BamHI, then I cut again with NheI because this cuts in the insert I want to replace to make self-ligation more difficult, after this I defosforylate, I tried AAP, CIP and SAP, and I ligate a PCR-product also cut with BamHI and EcoRI and in my primers there are always 8 bp next to the primer site...).
(the restriction analysis is not explained by ligation of Bam-NdeI of EcoRI-NdeI or any of that kind, I've checked all the possibilities).
I'm thinking of trying out SURE cells, of Stbl2 cells as I think maybe my cells do some recombination, but am not sure if they are really that much better.
Any suggestion welcome, thanx in advance.
do you run the fragments out on a gel and then gel purify them? because if you don't, that's almost certainly your problem.
if a positive control is going into the cells each time, then the cells are unlikely to be the problem.
but try XL-1 blue chemical competent cells if the DH5alpha just won't work. they're quite reliable.
I have tried all possible combinations of gel purifying my vector and also my insert, and then combining with the other gel purified and not.
I have even tried cutting my ligation reaction with an enzyme that only cuts in my "old insert" and not in the one I want in there.
The only thing left trying is cloning my insert in a Topo-vector and then cutting it out (by this I am sure that my restriction enzymes have indeed cut my insert, which by cutting the PCR product is of course not the case, but not cutting of my PCR product does not explain the digestion results afterwards).
This is what I usually do when I amplify a fragment by PCR, because TA ligation depends little on insert sequences.
I gel purify the insert before ligation.