Genomic library in pZErO

Gerd gerdn at ibg.uit.no
Tue Dec 8 05:53:50 EST 1998


Hei
If the cost of competent cells is your only limitation, why not just
make your own electrocompetent cells, it's very easy (follow protocol
that comes with your electrophoretic apparatus, or look it up in any
laboratory manual), takes not very much time and should save you some
money. Then reuse the electrophoretic cuvettes (you , which saves you
even more money. Now it's just the timeconsume that still will be high.
But I think shopping around for other procedures also takes some time -
so you will have to make a choice.

Gerd

> We then tried the recommended strain for pZErO,  Top10 which
> constitutively expresses the ccdB lethal gene without the addition of
> IPTG.   The highest competency available in these cells are the
> electrocompetent Top10s (109cfu/ug) which are about $60/aliquot which gave
> me 10/10 transformants with inserts but at best I could get 5000 colonies
> per 80ul aliquot of cells.  I need approx
> 150 000 colonies in my library, so this seems a very expensive way to go.
> 
> Any comments or suggestions/references would be appreciated
> 
> Thanks,
> 
> Kristina.



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