cloning problem

converrl at converrl at
Fri Nov 25 14:09:54 EST 1994

     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)

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



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