**HELP!** Binary Vector Ligations

[Alexander Kraev]

>1200bp into the plant binary vector pBI121 (Clontech) with the intention of
eventually transforming into Arabidopsis. The vector is approx. 13 kbp in
size, and we are using a simple EcoRI/Hind III digest, so there should be no
problems.<

I got the same problem with ligations of  2 kb fragments into a binary
vector that was 16 kb long. I think the problem is actually two-fold:
first you need to achieve a nearly complete digest, and since it is rarely possible, I always gel purify the vector, however, with long plasmids one should be patient and run the gel overnight, so as to separate the linear from nicked circular form very well; second, your competent cells have to be better than 10E7 per ug of pUC18. You are much better off with electroporation or Inoue chemical protocol, than with a simple CaCl2.

I hope this helps,


Alexander Kraev, PhD                     Internet:    kraev at bc.biol.ethz.ch
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