Yeast Spheroplast Protocols

Klaus.Matthaei at ANU.EDU.AU Klaus.Matthaei at ANU.EDU.AU
Mon Jun 27 03:04:54 EST 1994


27-6-94

Dear Netters,
        I am writing on behalf of a colleague who has turned his hand to
protein expression using the Pichia Yeast System from Invitrogen. In
particular the query relates to formation of spheroplasts and subseqent
transformation with the recombinant plasmids.
        He has tried the "standard" protocol(in the Invitrogen Pichia
manual), for spheroplast formation, of sorbitol washes, Zymolase treatment
then PEG/CaT transformation.This takes a long time (about 5-6 hrs, ends up
a long day etc., etc., etc....) and he was wondering if there are any
shortcuts or even shorter protocols. Or is it a case of practice makes
perfect?
        Also, which is the preferred method for spheroplast transformation:
PEG/CaT or Lithium Chloride or Electroporation?
        BIO101 seem to have a kit which makes spheroplasts in 1 hour. Has
anyone had experience with it or is it just pie-in-the-sky?
        Any comments or protocols would be greatly appreciated.

                                        Thanks    Klaus

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Dr Klaus Matthaei                               |           |
The John Curtin School of Medical Research      |  _--_|\   |
The Australian National University              | /      \  |
Snail mail: Canberra, ACT 0200, Australia       | \_.--._/<<|
E-mail: Klaus.Matthaei at anu.edu.au               |       v   |
Landline: 61 6 249 3782
                        "Sometimes I'm the Louisville slugger"
                        "Sometimes I'm the Ball"
                                Dire Straits
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