Cloning problems

[Roland Hubner]

> i am trying to make vectors (expression, binary) with my gene
> however:
> 
> with a blunt-ended insert -> only  colonies with fragment in the
> antisense direction
> with         BAMHI-insert->  only  colonies with fragment in the
> antisense direction
> 
> with SpeI-BAMHI (supposed to be directional) -> colonies but empty, but
> when PCRing the ligation there is something directional...
> 
> I believe that there is something toxic for the bugs....
> Any hints how to get around this really annoying problem


Hello,

 1/ cut with BamHI and re-ligate, screen for sense: just to make sure 
that toxicity is the culprit...

 2/ for bacterial expression there are numerous TIGHT promoter 
expression systems...

(e.g., albeit for these you need license: "Pl", see LaVallie et al. 
(1992) Bio/Technology 11:187-193 --  Biogene, Inc.; "araBAD", see Guzman 
et al. (1995) J. Bacteriol. 177:4121-4130 -- Xoma, Inc.)

 R.