Pichia problems

R. Jayakumar jakku at mrna.tn.nic.in
Tue Jun 19 07:59:40 EST 2001

How do you do your subcloning... ??

----- Original Message -----
From: ""W. Stuart Annan"" <wsa at st-andrews.ac.uk>
To: <methods at hgmp.mrc.ac.uk>
Sent: Tuesday, June 19, 2001 3:53 PM
Subject: Pichia problems

> 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
> ---


More information about the Methods mailing list