cloning problem

Klaus Salger salger at wap18.zi.biologie.uni-muenchen.de
Fri Nov 25 18:41:26 EST 1994


converrl at ucbeh.san.uc.edu wrote:
:      I had a question about a cloning step I am trying to complete:

: 1. I want to ligate a 2.5 kb BsaBI/KpnI fragment to a pBluescript vector
: containing a 9.6 bp insert which has also been cut with BsaBI/KpnI.

: 2. I have had no difficulty in unidirectional cloning like this unless it
: involves blunt ends (which BsaBI produces). When I attempted this ligation
:, I
: got back very few colonies, and the ones I checked were all edited (smaller
: than the desired construct).

: 3. I am particularly worried about this ligation, since BsaBI not only
: produces
: blunt ends, but also has to cut at 60 degrees Centigrade. My feeling is
: that
: cutting at high tempratures like this may have a tendancy to knock
: phosphate
: groups off the end of the fragment, and thereby leave more than the usual
: number of knicks in this ligation product. Since knicks are great sources of
: recombination, I am worried that this may be the primary source of all this
: editing activity.

: 4. The strain I am using is XL-1 Blue. I was wondering if SURE cells might
: be a
: better choice to avoid all this editing.

: 5. If my hypothesis in #3 is correct, I thought I might be able to fix the
: problem by using polynucleotide kinase on the fragments prior (or during)
: ligation.

:      I would appreciate any information on these points--please e-mail any
: helpful suggestions!!

:                        Thanx--

:                        Richard

Richard,
I had a similar problem some time ago which turned out to be a
nuclease contamination in the ligase buffer. So, if you didn't check
this yet, I would recommend to do a test-ligation with something else.
If the ligation itself isn't the problem, maybe the resulting construct
is expressed and toxic for E.coli. In this case you could try to use
another strain which maintains a lower copy number of the transformed
plasmids (e.g. ABLE from Stratagene).
BTW, I didn't notice a higher percentage of junk clones when using
dephosphorylated vectors (I also use XL1 Blue).

Cheers
  Klaus

--
Klaus Salger                phone : ++49 (0)89 5902 -502
Zoologisches Institut       FAX   :                 -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
Luisenstr. 14
80333 Muenchen
Germany



More information about the Methods mailing list