Problems with cytoduction

Ronan O'Kennedy Ronan.okennedy at FRMMIC.UCG.IE
Wed Mar 6 12:50:14 EST 1996


I have a problem with cytoduction. I am trying to introduce an episomal
expression vector (pLG669-z >> Ura+ Bgal+) into a range (about 60) very
closely related strains of S.cerevisiae (GC379 typ his leu ura) . The donor
YPH925 pLG669-z ( lys ade trp his leu cyh kar1-1) was mated with the GC379
derived strains by mixing donor and recipient in YPD and growing for
12-16hrs.Cells were washed and plated to select for recipient with plasmid
on WDm dropout -lys -ura and then replica plated onto WDM-selective again.

The plasmid stability of the cytoductants which display the correct markers
is then determined. The problem is that after an initial 2 rounds of
selective growth the cytoductants (of the original strain or derived
strains) exhibit extremly slow or no growth on the selective plate and
broths. I would expect that the cytoductants of these derivatives (which
grow normally) should exibit similar growth characteristics (assuming a
slight (10%) growth rate reduction due to plasmid carraige) and not the
60-70% growth rate reduction that we are observing. Can anybody figure what
is causing this massive growth rate reduction?

Is there something that I am not taking into account?

Ronan

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Ronan O'Kennedy                         Email Ronan.okennedy at ucg.ie   OR
Fermentation Lab                              Ronan.okennedy at frmmic.ucg.ie
Dept of Microbiology
University College Galway
Ireland
Voice 091-524411-ext 2191
Fax   091-525700                 http://frmmic.ucg.ie/labwww/Ronan/Ronan.html
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