help needed with three fragment ligation

Chris LaRosa clarosa at biocomp.unl.edu
Wed May 10 10:14:31 EST 2000



Colin Herd wrote:
> 
> Is there anybody out there who may be able to help me? (don't worry this
> is a question about molecular biology, I go to, alt.rec.personalprobs,
> for other types of help:-)
>     I have a puzzle with regard to a ligation I am trying to perform. I
> want to ligate three pieces of DNA together, one of 7.1kb, one of 3.6kb
> and one of 1.9kb, to form my complete plasmid for bacterial
> transformation.All the fragments use different sticky ends to minimise
> problems with the pieces religating in the wrong orientation/order but
> it doesn't seem to help. I have done the reaction a number of times
> without success.
> 
Can you break it down to two sets of ligations....1 to 1 , then 1 to
one..?

I have not tried 3 frag ligation very often...but when I did it never
worked.




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