blunt ligation problems

Jim Graham graham at BIODEC.WUSTL.EDU
Thu Aug 27 15:08:45 EST 1998


Aye,

30% of DNA ends would mean something like half have at least one end with
a non-templated addition. That leaves half the molecules with two
ligatable blunt ends, and somwhere less than 30% available for ligation to
a vector having T overhangs.

No explanation has been offered for why current strategies succeed in
going after the minority population while simple kinasing and blunt
ligation would be the obvious route.

Jim
J. Graham PhD 
Biology Department 



On Thu, 27 Aug 1998, Patrick F.H. Lai the Graduate Student wrote:

> ---- Begin Original Message ----
> > If you used Taq to make the PCR products, they will leave hanging A's
> > are NOT blunt product.. you would have to digest
> 
> Oh, but some 70% of the ends generated by Taq polymerase are indeed
> blunt. The basis for their greatly reduced ligatability has not been
> adequately explained. (A general reduction in ligation efficiency
> following PCR is widespread across all sorts of different techniques
> which try to get around the basic observation. This has been an
> unresolved problem with PCR from day one.)
> 
> Anyone?
> 
> (E-mail copies pleeeease)
> 
> Jim
> J. Graham PhD
> Biology Department
> ---- End Original Message ----
> 
> Dear Dr. Graham,
> 
> i don't know how the '70%' was determined (i heard this from many
> people, so it must be true....), but i thought it only takes one end
> to have the 3'-overhang to mess up a ligation to insert the fragment
> into a vector with perfectly blunt ends.
> Hence, ligatability Taq-generated PCR products is so low.
> 
> One may also suspect other modifications at the 3'-ends by Taq.
> Whatever.....
> TA-cloning works, and I AM HAPPY !!!!!   8-D
> 
> 
> 
> 
> 
> Hope this is useful info.   :-)
> 
> 
> Patrick F.H. Lai  < PFHLai at looksmart.com >
> Graduate Student
> University of Toronto
> Toronto, Ontario, Canada
> 
> 
> 
> 
> 
> 
> LookSmart … or keep looking.
> http://www.looksmart.com
> 




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