pGEX-4T Expression Vector Problems

Tony Hodge tph at mrc-lmb.cam.ac.uk
Thu Mar 17 03:45:12 EST 1994


I have been trying to clone some cDNA fragments into the pGEX-4T
series of plasmids (Pharmacia) with little success.  The main
problem seems to be a low transformation efficiency into E. coli
JM101 and BL21 using either Ca competent cells or DMSO treated
cells.  Typical efficiencies seem to be 1E3 to 1E4 per microgram of
DNA (pGEX-4T-1 and pGEX-4T-3) direct from the tube supplied, let
alone after cutting/re-ligating.  (As a control Bluescript plasmids
give 1E6+ per microgram with Ca competent cells without fail.)

Has anyone else had similar problems?  Are other hosts or competent
cell prep methods better?  Pharmacia have not yet come up with any
alternative suggestions  : - (

Why do I want to use pGEX you may ask in the light of my problems,
well one of my colleagues has a clone obtained from another lab
which expresses very :-) well indeed.



Tony




.
Tony P Hodge
Structural Studies Division
Medical Research Council Laboratory of Molecular Biology
Hills Road
Cambridge
CB2  2QH
UK

Phone (0223)  402260

Fax     (0223)  213556



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