Genomic library in pZErO

Kristina Mills krism at ichr.uwa.edu.au
Tue Dec 8 04:07:12 EST 1998


I have been trying to construct a genomic library in the pZErO plasmid
vector as I thought this would be a quick, easy and inexpensive way to do
it.  I have however had some problems.  I digested genomic DNA in two
pools with Xho1 and Sal1 and size fractionated on a sucrose gradient for
5-20kb fragments.  These were ligated with Xho1 digested pZErO.  Initial
transformation attempts were made with XL-10 Gold ultracompetent cells
from Stratagene which report a transformation efficiency of 5 * 109/ug
with large inserts. IPTG was used in the plates to induce expression of
the lethal gene at the recommended concentration,1mM  (10mM IPTG was also
tried).  This resulted in millions of colonies with at best 1 out of 20
with an insert.  Replica plating of these colonies onto plates containing
IPTG resulted in the death of those colonies without an insert.  We
concluded that the induction of the death gene in this strain of cells was
not working properly.  Why?

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.

-- 
Kristina Mills

TVW Telethon Institute for Child Health Research
(Company Limited by Guarantee)
Western Australia

Fax 61 9 388 3414



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