Ligation problems

brett at BORCIM.WUSTL.EDU brett at BORCIM.WUSTL.EDU
Sat Mar 11 10:38:16 EST 1995


>Dear bionetters,
>
>I presently have big ligation problems but that was expected. I need to
>put a 3kb insert into a 3.2 kb vector. Oh, did I tell you it is a blunt
>ligation :-0
>
>I tried treating the vector with alkaline phosphatase but the ligation
>effeciency is so low I didn't get anything.
>
>If anybody have a knock-out technique for hard to do ligation, I would be
>very grateful.
>
>
>Francois Nantel
>IGBMC
>nantel at titus.u-strasbg.fr

why was this expected? there could be a number of reasons why it didn't work,
least among them the sizes of your fragments. try to address the following,
experimentally:
        1. You did not kill your AP, which can inhibit ligation.
        2. What are the conditions of your ends? Can you ligate the
           un-AP'd vector back together? Can you ligate in another
           blunt end fragment (smaller, if you choose)?
        3. If the ligation efficiency is low, transform highly competent
           hosts, so you can find it.
        4. Optimize the conditions for blunt end ligation (use the un-AP'd
           vector) in your hands.
        5. Vary the insert:vector ratio used. Most people seldom have the
           [DNA] they think they do, following all the manipulations you
           probably did to get the fragments.
After you try all these write back & let us know how it turns out.
Brett Lindenbach
Lucille P. Markey Student in Human Pathobiology
Program in Immunology
Washington University - St Louis
brett at borcim.wustl.edu





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