Problems with One Shot INValphaF' from Invitrogen

sunfish s535290 at aix1.uottawa.ca
Wed Feb 12 13:01:16 EST 1997


> We have had exactly the same problem.  All of a sudden I became the man
> who could not clone.  Invitrogen have replaced the cells (with more
> clear ones) but everything still sucks.  So if anyone from Invitrogen is
> out there, please change or we will!
> 
> Martin

Martin,

just to follow up on my initial post, I have pretty convincing evidence
that the problem is the transformation efficiency. When you try to
transform a tube of cells with 10 pg of supercoiled pUC18, you'll be lucky
to get even one colony. Boost the amount of DNA used to 1 ng of pUC18 and
then you start seeing transformants. A drop in tranmsformation efficiency
of 2 orders of magnitude. If get 100-200 transformants per TA cloning,
you're not likely to get many transformants now. Side by side comparisons
with home made cells using the TSS protocol gave us more transformants
using our homemade competent cells. I have gone back and transformed some
of the ligations that had previously yielded nothing with their cells,
plated them out only to get a decent number of transformants. Since the
strength of their cells is supposed to be the fact that they're more
competent, the product is clearly not meeting QC. I have never had
problems with them before. In fact, until a couple of months ago, I would
have sworn by the TA cloning kit. Seeing other struggle with homemade
kits, or blunt end cloning while we could routinely clone any PCR product
we wanted had me convinced there was no other way to go if you wanted to
get results quickly. But I'm not going to shell out all the cash for a kit
for which a hugely inportant (and expensive) component is of below
standard quality. Should we just start making our own T-vector ?

Ed




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