Blut-ending ligation

[Michael Cooley]

Mr. J. Membrillo-Hernandez (jmembril at hgmp.mrc.ac.uk) wrote:

: 	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.
: 						Sian


Why do you use T4 poly in one place and Klenow in another? I use T4 poly 
for all my bluntings because I think it is more reliable. But you had 
better do the reaction at 12 oC; the 3' to 5' exo activity is too strong 
at higher temps. Just use the reaction conditions in Maniatis. 

For blunt ligations I use a 22 oC reaction temperature with standard 
buffer and reagent concentrations overnight.


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