seemingly easy ligations don't work - (Sep/19/2005 )
Well I was too quick... in fact I had three colonies the last time I tried, two for one construct and one for the other.... I grew them o/n yesterday, did the mini's and the digestion... and it turns out all three are good (I had none on the control plates...)
Still there is a major problem here with the transformations, we've tried chemically competent cells yesterday with some controls... nealry 50 times more linearized, religated plasmid than linearized alone. I guess we'll keep the home-made competent cells for intact plasmids (work fine) and either make a new batch for ligation experiments (it was the first time I made competent cells, so I might have done some things which are getting the efficiency down, even if I did everything on ice in the cold room) or buy cells.... because the subcloning efficient cells we bought now are not very expensive, certainly not in comparison with the time I lost by trying and retrying
Do some of you have little tricks of what to do to be sure to have good competent cells ?
Make absolutely sure that there is no detergent of any kind anywhere near the cells or media. Think about media prep and growing in fresh plastic rather than washed glass, e.g., or rinse the glass very very well.
We learned very recently that the small amounts of Triton-X100 carried over from the EcoRI restriction digest buffer, into the ligation mix, and then into the competent cells for transformation has a dramatic (100x - 1000x) negative effect on the transformation rate.