Blunt end ligation
Morris F. Manolson
mm6y+ at andrew.cmu.edu
Thu Feb 25 15:41:35 EST 1993
> Can someone please send me a copy of their protocol for blunt end
>ligation of DNA fragments. Notes on pitfalls would be most appreciated.
I do my blunt end ligations almost the same way I do my sticky
end ligations except for
1) I phosphatase the vector at 50 C (instead of 37 C) and use twice the
units of Calf Alkaline Phosphatase.
2) I do the ligation at Rm. Temp (instead of 12 C).
3) I up the ratio of insert to vector from about 2:1 to 4:1
If I am having a real hard time, I will first do the ligation
in 10 ul for a couple of hours at Rm. Temp. (or untill I am ready to go home)
bring the volume up to 19 ul with buffer, add another ul of
ligase and let it sit o/n at 12 C.
Apparently Calf Alk Phos has alot harder time getting the
5' phosphate off a blunt end rather than a 5' sticky end.
If you have lots of vector religation, this may be your problem.
hope this helps, Morrie
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