pcr from yeast? (2 hybrid question)

jfh jfhess at ucdavis.edu
Wed Feb 7 15:03:19 EST 1996


Howdy.

I am doing some yeast 2 hybrid system screenings.  I am not a yeast
person but things are going OK.  I have made constructs and put them into
yeast strains.  I have the control plasmids sold by clontech.

To absolutely verify that the yeast conatin the constructs I think they
do, I'd like to pcr acorss the vector and insert boundary.  I have oligos
which I used to sequence the constucts to verify in frame ligations and
oligos specific to my proteins.  However, PCRing from whole yeast doesn't
work.  I have experience with PCRing Ecoli,  Lambda phage plugs and
mammalian genomic DNA.

Can someone give me a way to PCR from whole yeast?

I did a miniprepof plasmids from yeats and transformed Ecoli and then
used that to verify the yeast had the plasmid I thought they did but
that's a lot of work for each construct.  Plus, if a yeast is harboring
two plasmids that each have only amp selection in ecoli, you have to
check to see what plasmid your ecoli got to make it amp resistant.


Thanks,

john hess  jfhess at ucdavis.edu



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