cloning problem...

Chris Boyd cboyd at holyrood.ed.ac.uk
Wed Aug 2 10:40:51 EST 2000


"Dr. Roland Barten" <rb248 at mole.bio.cam.ac.uk> wrote:
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: 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?

It does sound like toxic protein expression.  A way round this is to
insert an intron into your construct (assuming you're talking about cDNA
cloning, and that your eventual aim is to look at expression in
eukaryotic cells).  If you were clever with primer design and lucky with
sites, you might be able to PCR clone a mini-intron into your stable
2-bp mutant construct and reverse the mutation at the same time.  Of
course this is more likely to work if the intron is near the 5' end of
the gene.  We took this approach with CFTR with some success, see:

   Boyd, A. C., Popp, F., Michaelis, U., Davidson, H., Davidson-Smith,
   H., Doherty, A., McLachlan, G., Porteous, D. J. and Seeber, S. (1999)
   `Insertion of natural intron 6a-6b into a human cDNA-derived gene
   therapy vector for cystic fibrosis improves plasmid stability and
   permits facile RNA/DNA discrimination.' J. Gene Med., 1, 312-321.

Best wishes,
-- 
Chris Boyd                      | from, but \    Medical Genetics Section
Chris.Boyd at ed.ac.uk             | not for,  /    MMC, Edinburgh Uni.,
http://www.ed.ac.uk/~cboyd                       EH4 2XU, SCOTLAND






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