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homologous recombination

Cathelijne van Duren Cathelijne.van_Duren at molmed.mas.lu.se
Tue Apr 25 12:06:43 EST 2000


I have a question regarding homologous recombuination in yeast.
I have a yeast strain inwhich I need to delete/inactivate the TRP1 gene.

I have amplified up the TRP1 gene with primers having 2 point mutations
delivering the TRP1 gene out of frame.

I then transform the yeast cells using LiAc according to Gietz method
with the TRP1- gene (PCR product) and a plasmid ensuring that I only
screen cells which have the potential of  taking up DNA.
The cells are grown on SD+Ta nd then replica plated onto SD-T
 cells are scored that do not grow on SD-T but do on SD+T.
I have had no luck sofar and I have screened a few miljon.

Can someone please give me some advice for increasiing the probability
of recombination occuring. I was told I have a change of less than 1 in
miljon, is this figure correct?



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