pEGFP-C1 transformation

Carnac carnac at realitys-edge.com
Sun Dec 22 06:23:01 EST 2002


Hi, Duncan, thanks for your reply.  :)

"Duncan Clark" <junk@[127.0.0.1]> wrote in message
news:QmasIUcA5ZB+EAIx@[127.0.0.1]...

> So is the problem how you treat the ligation mix prior to
> electroporation?

Presumably... either that, or for some bizarre reason, the electroporation
is frying the cells, which would would seem less likely.  The controls
fried, yet the vector/insert did not arc, which seems unusual, as the
vector/insert would presumably contain more salt contamination... it is
pretty unlikely that the two were mixed up, but there is always that
possibility...

> Ligase kills the electroporation unless you heat kill it and as I
> electroporate using my home made device, which uses around 300V but much
> smaller gaps (0.025mm), I get rid of all salt. The dextran blue is just
> a nice carrier and doesn't hurt anything.

Ah, this is interesting... I didn't actually heat kill the ligase.  Do you
think this is the most likely possibility for the poor transformations?  I
will give this a try.  Thanks.  :)






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