Pichia problems

[Maxim Rossokha]

"W. Stuart Annan" wrote:
> 
> Hi,
> 
> I'm having problems subcloning into the pichia vector pPICZa via
> EcoRI/XbaI. Normally this would be an easy task but four attempts later
> I'm getting nowhere very quickly! I'm using XL1-Blue cells from Stratagene
> (these are competent) and my DNA ligase does work. I'm using Zeocin at
> 25ug/ml in my low salt agar plates but the odd colony which does grow on
> them takes 24-48 hours to grow up in broth and then the miniprep doesn't
> give me any DNA (not even religated vector!). Also, can anyone suggest an
> alternative to Zeocin from Invitrogen as it's a tad expensive!
> 
> Stuart Annan
> 
> ---

Hi!
May be you have to check what kind of resistance genes are in your
plasmid.
I think it is Ampicillin. You have to grow your transformants E.coli on
ampi-plates and not on Zeocin.
After the transformation in P.Pastoris your gene will be integrated in
the genom!! Your transformated yeast will get a Zeocin resistance!!

Maxim Rossokha