help needed with three fragment ligation

Ian A. York iayork at panix.com
Wed May 10 10:32:34 EST 2000


In article <39197CD7.21B1B328 at biocomp.unl.edu>,
Chris LaRosa  <clarosa at biocomp.unl.edu> wrote:
>Colin Herd wrote:
>> 
>>     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
>> 
>Can you break it down to two sets of ligations....1 to 1 , then 1 to
>one..?

Agreed.  Three-fragment ligations sometimes work, but more often they
don't; it's not worth fooling around with it, if it doesn't work after the
first couple tries.

If you don't have the right ends, use PCR, with a proof-reading enzyme,
to put the right ends on.  Or subclone into a multiple cloning site and
pick up the ends you need.  Even if you have to do two or three extra
clonings, it'll probably still be faster than making the three-fragment
work.

Ian 
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England




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