speI xbaI sublconing

Aawara Chowdhury via methods%40net.bio.net (by aawara from FEMA-trailer.org)
Fri Dec 7 22:52:15 EST 2007


In <vuy7ijqp4j1.fsf from shaggy.csail.mit.edu>,
 Tom Knight <tk from csail.mit.edu> wrote:

> "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.

SpeI and XbaI have compatible overhangs, and uni-molecular reactions are
always favored over bi-molecular reactions.

Unless the original poster dephosphorylates the ends of his cut vector,
it will recircularize very efficiently, and none of his clones will contain
the insert.

AC
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