Ligation problems

Vural, Ali via methods%40net.bio.net (by AVural At lsuhsc.edu)
Fri Dec 8 13:55:48 EST 2006


Hi, 

 

I just read your answer to ligation problems.
 
 
 "It is possible that for
some strange reason some polymerase is getting carried over into your
ligation reaction, and in the absence of nucleotides in ligation buffer
(with the exceprion of ATP), the polymerase chews back on the the Watson
strand. Thus, you are getting all possible combinations of correct
sticky
ends of the vector, as well as blunt ends, or reverse sticky ends.
Another
solution (and the way to prove that I am right) would be to supplement
the
ligation buffer with all four nucleotides. BTW, you could even try
adding
only CTP and ATP in your ligation. Could you do that and post with the
results?
Cheers,
Emir

 

 

Can you please explain methe molecular basis of chewing back of Watson
strand and getting the possible combinations of DNA fragments?

 

Thank you,

Ali



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