Ligation problems

Klaus.Matthaei at ANU.EDU.AU Klaus.Matthaei at ANU.EDU.AU
Fri Mar 17 18:49:52 EST 1995


>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                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


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.

I have used this system also with some success but the enzyme is extremely
expensive more than $A1 a unit here in Australia and only one manufacturer.
We also tried to use SmaI to cut in the internal sequence of the 8 base
cutter to open up the vector to try to save on enzyme.  This was worse for
the system since SmaI can and often nibbles the blunt ends and does not
allow blunt ligation.  Apart from that it does work but as I say is
expensive.  We also have a pGem7 vector that also has this site engineered
if the manufactures vector does not have usable sites for further
manipulation.

Cheers, Another Klaus

*************************************************************************
Klaus Matthaei
Head, Gene Targeting                            |           |
The John Curtin School of Medical Research      |  _--_|\   |
The Australian National University              | /      \  |
Snail mail: Canberra, ACT 0200, Australia       | \_.--._/<<|
E-mail: Klaus.Matthaei at anu.edu.au               |       v   |
Landline: 61 6 249 3782 fx: 61 6 249 0415
                        "Sometimes I'm the Louisville slugger"
                        "Sometimes I'm the Ball"
                                Dire Straits
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