speI xbaI sublconing

Tom Knight via methods%40net.bio.net (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.

Two options:
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
   vice-versa.



More information about the Methods mailing list