speI xbaI sublconing

Michael J. Prigge via methods%40net.bio.net (by mprigge from indiana.edu)
Fri Dec 7 20:19:17 EST 2007


You didn't mention any problems with the digestion, but I thought I  
would throw in this reminder, just in case.  XbaI is susceptible to  
dam methylation, so TCTAGAtc and gaTCTAGA will not be cut.  If this  
isn't the problem, I would try Tom's options, possibly screening  
colonies by PCR with an insert primer and a vector primer for the  
correct orientation.
Good Luck,
Mike

>> "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.
>>
> Tom Knight via methods%40net.bio.net (by tk from csail.mit.edu)
> 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.
>
>








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