cloning problem...

[Dr. Hiranya S. Roychowdhury]

This, IMO, is a classic case of an expressed foreign protein that is toxic
to E. coli (especially in light of the fact that a 2-bp change allowed the
plasmid to be maintained). I see no way of circumventing this problem
except through the use of a non expressing vector. Although the TA vectors
make life somewhat easy, cloning polished-off PCR products into blunt sites
is no great hassle. The prokaryotic promoter used in BW selection would
prove counterproductive in such cases, hence a promoterless vector may be
useful. However, to begin with, you may try foregoing BW selection (keeping
off the IPTG may keep the promoter inactive enough)

At 10:00 AM 7/31/00 +0100, Dr. Roland Barten wrote:
>We have been trying to clone a human gene in to TA vector. We've got
>blue/white selection,  have screened the white ones directly by PCR, and
>then tried to grow up those with inserts - this is where the problem
>starts. On trying to grow up for mini-preps, nothing happens. There is
>absolutley no growth.
>We have tried different media and both Kan and Amp selections (as well
>as no antibiotics at all), but we just cannot get any of these colonies
>to grow in liquid media - except for once when it turned out that the
>insert had two base pair changes.
>One option would be phage - any other suggestions?
>
>Thanks
>Anne-Marie Lucas
>
>Attachment Converted: "c:\eudora\attach\vcard.vcf"
>
Dr. Hiranya Sankar Roychowdhury
Dept. of Molecular Biology	
PO Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu


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