speI xbaI sublconing
(by tk from csail.mit.edu)
Fri Dec 7 16:40:34 EST 2007
"V Arunachalam" <vadivelarunachalam from yahoo.com> skrev i melding
news:mailman.782.1196360538.23109.methods from net.bio.net...
> I am facing problems in cohesive subcloning a 500 bp insert from the
> double digest of SpeI and XbaI onto a vector of 13.5 Kb size at the same
> sites. Please suggest me is it correct strategy as these two enzymes
> generate similar ends though differ in recognition sites.
1) Ignore the problem and select one of the two orientations after the
fact. About half should be in the orientation you want.
2) Add SpeI or XbaI to the ligation mix. This will force the ligation
which recreates the SpeI or XbaI site to be cut, favoring the
ligation of the vector SpeI site with the insert XbaI site, and
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