Blut-ending ligation

Mr. J. Membrillo-Hernandez jmembril at
Fri Jan 27 10:35:52 EST 1995

	I would like to clone Tc gene (from pBR322) into a fragment of 
500bp (to interrupt it), I have tried several times with out success, the 
main problem is no compatible ends so I have to do a blunt-ended 
ligation. Is it the enzyme I am using?.
	This blunt-end ligation is preceed by a T4 DNA polymerase filling 
of the vector in the site Sph1 (in the middle of my fragment) an the Tc 
gene was cut with the Ava1 and Ssp1 (I think this fragment contains all 
the fuctional Tc gene), then Tc fragment was blut ended with klenow.

	Could anybody help me to set:

	a) good conditions for klenow reaction
	b) good conditions for T4 polymerase
	c) good conditions for blunt end ligation using T4 DNA ligase.

Thanks in advance.

More information about the Methods mailing list