Pichia pains

[Beth Wapelhorst]

If it's your transformation, that's easy to check by transforming the
intact vector as a control. If that's okay, then somethin's wacked with
your ligation reaction. You didn't say what your insert frag was -PCR
product? -cut from another vector? Are you phosphatasing your vector? Can
you religate cut vector alone? It's them simple cloning jobs that get ya,
because you figure it'll just pop in the first time, no problemo, so you
get lazy and don't do your controls...then when it doesn't work, you have
no idea why. I do this all the time, too...you'd think I'd learn ;)

beth
DNA Hacker's Resource:
www.blarg.net/~weazel/DHR/dhr.htm

Michael O'Donohue <odonohue at lille.inra.fr> writes:

>Hi everyone!

>Does any have any experience with the larger Pichia vectors (eg pHILS1,
>pPIC9 etc.)? I am having a load of trouble trying to clone a fragment
>into the EcoR1 /BamH1 sites of pHILS1. Such a simple cloning job!!!! I
>have lots of vector, lots of fragment......but not even one miserable
>colony. Is transformation efficiency at the root of the problem?

>Any ideas?
-- 
sugar beth wapelhorst              "I don't think I'm alone when I say I'd
weazel at blarg.net                    like to see more and more planets fall
www.blarg.net/~weazel               under the ruthless domination of our
                                    solar system." - Jack Handey