Cloning problems-Help!!

[Andrew Leung]

How the insert fragment comes from? PCR or cut from other vector. If PCR, will it be too close to the end for any one of the restriction enzyme.

Andrew
    zimprich ¼¶¼g©ó¤å³¹ <3841655C.A7E7D861 at nefo.med.uni-muenchen.de>...
    Dear Colleagues, 
    In the last few months, i have a very big cloning problem,. 
    I try to get a AOC1 (5¡¦overhang CC!TGAGG), ECO R1 (also 5¡¦overhang G!AATTC) fragment into a  vector also cut with AOC1, ECO R1.  It sounds quite easy, but it is a nightmare. 
    I tried it over a dozen times, but i only get  very few colonies after transformation, which were all wrong. I did all kinds of control experiments; so let us assume that Ligation, Transformation etc. do work in my hands. 
    Is it possible the some restriction enzyme overhangs get lost, or need a special treatment for ligation? 
    I purify my DNAs over agarose gels and clean them up using Quiagen coloumns. 

    Any ideas 

    Thanks a lot 

    Alexander 
      

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