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