Ligation problems

Klaus Salger salger at wap18.zi.biologie.uni-muenchen.de
Sun Mar 19 16:27:57 EST 1995


Klaus.Matthaei at ANU.EDU.AU wrote:
: >wmbonner at helix.nih.gov wrote:
: ><snip>
: >: I've had good luck with the pCR-script kit from Stratagene.  It uses a
: >: rare 8-base cutter (I forget
: >: which) to recut the vector each time it closes without the insert.
: >:  No need to dephos. Good as long
: >: as your frag does not have the 8-base cutter site.
: >: -Bill
: >
: >The same does of course work with every enzyme, provided that its site
: >is unique in the vector and doesn't cut the insert.
: >
: >Cheers
: >  Klaus
: >
: >--
: >Klaus Salger


: Not True.

: The system only works with blunt cutters so that when an insert is ligated
: into the vector the original restriction site is not reconstituted.  If
: religation occurs without insert, the enzyme recuts the site.  With sticky
: restriction sites the site is obviously reconstituted and the enzyme cuts
: the insert out again.
<snip>

Oops, you're right. I have to add that the vector site must not be
reconstituted as is the case with different REs with compatible overhangs
or partially filled in sites.

Sorry
  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



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