help needed with three fragment ligation

George Rutherford gruther at bilbo.bio.purdue.edu
Thu May 11 13:59:46 EST 2000


In article <391A86C2.907D0E52 at uni-konstanz.de>,
Frank.Fackelmayer at uni-konstanz.de wrote:

> I agree with Ned that a three fragment ligation is not a problem
> usually. Of course, it depends on what you want to clone, and as always
> some framgents simply don´t clone well. If all fragment ends are
> non-identical, there is a good chance to get the right clone by just
> putting in all three fragments, ligase buffer and ligase. It may be
> necessary to check more minipreps than in a conventional two-fragment
> ligation, though. We routinely do 24 to 36 preps. 
> Good luck,
> Frank
> 

Three bits aren't usually a problem (excepting the occasional intractable
construct). I'll do 16 mps, and if that doesn't work, screening by colony
hyb almost always works and doesn't take very long either.

George




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