Mr. J. Membrillo-Hernandez
jmembril at hgmp.mrc.ac.uk
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.
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