oligo cloning

[ChenHA]

"R. John Lye" wrote:

> Caroline Szymeczek-Seay wrote:
> > Vector is cut with SmaI and CIPed.  Oligos are kinased.
>
> Be careful of over-CIAPing - in my experience, too much enzyme and/or
> too
> long a reaction will damage the ends of the DNA.  Make sure that all the
> CIAP has been removed, also.  I usually use a little SDS/proteinase-K
> digestion followed by phenol/chloroform to be sure it is all gone.
>

One useful protocol for blunt-end ligation without CIP:
http://mutant.lse.okstate.edu/chunming/onetube.html

but replace EcoRV with SmaI, pBluescript with pGL3 in this particular case.