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PEG and Blunt end ligation

Stefan Bäckström stefanba at bio.embnet.se
Fri Dec 15 14:18:55 EST 1995


I think you should ligate with T4-DNA ligase and cleave with sma I in the same
tube as described in BioTechniques vol 12 (1992) 28-30.

200 ng PCR product
50 ng plasmid (pBluescript in the article)
4 units T4 DNA ligase
5 units SmaI
ligase buffer, rATP, DTT as in a regular ligation (I asume, they dont tell)

in 20 µl

ligate/cleave in 22 degrees for 4 hours

Transform..

Since the SmaI site dissapears when you get an insert, the reaction will
be forced against insert ligation. Selfligated vector is cleaved and ready
for insert.

I haven´t tryed it myself, but Stratagene sells a kit based on this method
so I assume it works. They don´t use smaI though.

Good luck.

Stefan Bäckström



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