I have been having horrible luck with transforming the One Shot competent
cells that come with the TA cloning kit. We just got a new kit, and I
wasn't getting clones. I tested transformation efficiency with the pUC18
they supply and the efficiency was putrid. Transformation controls with home
made stuff were fine, so it really seems like it was the cells.
We just got a new batch of replacement cells, and much to my
horror and disappointment, the transformation efficiencies are still
stinkier than a bloated carcass rotting out in the sun (not that I'm
bitter or anything). I can get higher transformation efficiencies
with cells I made by the one step TSS method.
Invitrogen mentioned that the cells come in a new buffer which
apparently yields higher transformation efficiencies. But when I swirl the
cells around after the 1 Hr incubation at 37 degrees, I see *no* turbidity
at all. This would seem to indicate that their cell densities are very
low. They claim the lack of turbitidy is because the cells come in that
modified buffer. But I still think I should be able to see some cells when
I swirl the tube. When I overgrow the cells by leaving them at 37 for 2
hours, I definitely start seeing more cells surviving in selection plates.
This still doesn't help the efficiency problem.
Anyone having this problem ? Invitrogen have been very nice about
replacing the cells and all, but still, I can't get the cells to work
using their transformation protocol. Since a good 3rd of the cost of a TA
cloning kit is from the cost of the competent cells, I would like to get a
bit more bang for my buck. Just doing transformation controls has wasted
me quite a few tubes of cells. Enlighten me please.
Thanks in advance.
University of Ottawa