>> Hi,
>> Sorry to jump in, but you have been working for 5 months on the Pichia system.
> Have you obtained the correct transformants (selection of the yeast) and still
> not had expression? Your message is a little unclear as to how far you have
> gone. (There are probably a lot of us at the very early stages of setting
> inserts into plasmids who would like to know where the difficulties lie).
>> Bob Jackman
>> ------------------------------------------------------------------
> Robert W. Jackman *
> Beth Israel Hospital/ Pathology,Research North *
> 99 Brookline Avenue RN280B *
> Boston, Massachusetts 02215 *
>>jackman at sprcore.bih.harvard.edu *
>> fax (617) 735-3591 *
> ------------------------------------------------------------------
>>
Hi Bob,
Sorry to have been so vague, but I was in a bit of a hurry. I have had no
problem at all in ligating my insert into the yeast vectors. That's
extremely easy (ha ha). I have even had my recombinant vector sequenced to
make absolutely sure that the insert was where it was supposed to be and
in the correct orientation. My trouble comes when I transform the yeast.
I am getting very few recombinant colonies when I plate the yeast on the
RDB plates. Most of the time I get NO colonies. My viability controls
work very well so I know the spheroplasting is working okay.
The few times that I have gotten recombinant colonies, they do not express
the protein like they should. I take the colonies from the RDB plates and
streak them onto MM and MD plates. Then I take the colonies that grow
slowly on the MM plates but quickly on the MD plates and use these to
induce secretion of protein. As I said in my previous letter, NO LUCK SO
FAR!!!!!
One quick word of advice if you are just beginning to the use the kit.
When you grow up your yeast cultures at the beginning of the protocol, it
is a good idea to confirm that you are growing yeast and not bacteria (a
microscope works great). Bacteria love the YPD media even more than the yeast
do. I spent several weeks trying to transform bacteria before I discovered
this.
Jeff Temple(jstemple at whale.st.usm.edu)
University of Southern Mississippi
