Blund ends cloning

Alex Chang lpss at unixg.ubc.ca
Tue Dec 31 00:57:37 EST 1996


Greetings everyone,

I am trying to clone a Vent DNA polymerase amplified PCR product in to a 
vector which was cutted blunt by restriction enzymes.

It didn't work, of course. Since the primers used for PCR were not 
phosphorylated at 5' ends. Instead, the restriction analysis of the 
transformed colonies showed that the vector ligated to themselves.

So, my question is, first, whether anyone has some strategy to clone 
blunt end insert with 5'OH successfully. 

Second, if the insert has to be phosphorylated at the 5' ends by T4 
polynucleotide kinase, is that ok to use T4 DNA ligase buffer to do it. 
The reason I am asking is that it is holiday season now, what I have is 
only the T4 kinase but not the buffer. I found the T4 DNA ligase buffer 
(Gibco/BRL) is very close, except there are 5% PEG-8000 and use DTT 
instead of ME.

Happy Holidays   

--
Alex Chang
Pathology
University of British Columbia
achang at hivnet.ubc.ca



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