Pichia problems

W. Stuart Annan wsa at st-andrews.ac.uk
Tue Jun 19 05:23:41 EST 2001


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

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