speI xbaI sublconing

[Dag Rune Gjellesvik]

"V Arunachalam" <vadivelarunachalam from yahoo.com> skrev i melding 
news:mailman.782.1196360538.23109.methods from net.bio.net...
> Hi,
> 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.
>
>
> 
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Hello,

I understand - you cannot dephosphorylate both vector and insert, and both 
may circularize since they have compatible ends. I have been in the same 
situation. I suggest that if you can cut your vector with only one of the 
enzymes, lets say SphI, and dephosphorylate your insert. If you do the 
ligation in the presence of SphI restriction enzyme, you will prevent your 
vector from circularizing. However, the XbaI end of your insert may ligate 
to either end of the SphI cut vector. After a period for ligation, 
inactivate the SphI enzyme by heat treatment, and add more ligase to allow 
your construct to close (circularize). You may have to screen for correctly 
oriented inserts, if that matters.

I hope this will work,

Dag Rune Gjellesvik