Ligation of fragment with both blunt and cohesive end

Helen McBride helen_mcbride at hlthsci.med.utah.edu
Thu Jan 16 16:09:36 EST 1997


In article <32D639FA.11CA at sheffield.ac.uk> , R.E.Hough at Sheffield.ac.uk
writes:

>1. Do I need to purify the DNA following fill-in/trimming before I cut 
>with the second enzyme (BamHI) or will heat-treatment (75!C for 10 
>mins) be sufficient to inactivate the fill-in/trimming enzyme so that 
>it doesn't blunt the BamHI cut? 

Yes, you need to get rid of the other enzymes before treating with the
second enzyme or you'll treat the new cut as well as the old one. A
phenol/chloroform extraction followed by an ethanol precipitation usually
works well for me. There's no need to gel isolate unless you really want
to.
>
>2. To ligate types of both ends at the same time by do you (a) use the 
>amount of ligase that would be used for a routine blunt-end ligation 
>where all the ends are blunt, and (b) do the ligation at 14-15!C as 
>for cohesive-end ligation?
>

You treat a sticky-blunt like a blunt.

Good Luck.
Helen McBride
University of Utah 
Grad Student
Dept. of Onc. Sci.
helen.mcbride at genetics.utah.edu


"Where the telescope ends, the microscope begins, which of the two has
the grander view?" Victor Hugo



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