Hi all I need to place a gene under the control of a galactose promoter in the yeast genome. The gene I need to place under the control of the gal promoter will also be a selection marker gene. What's the best vector to use to do this?
Thanks for your time
Ed
I would suggest against using a vector for this. Especially if the gene you are inserting can be selected. Yeast is very good in recombination so all you have to do is give it the fragments with homology to each other and the target locus you wish to integrate in. Example fragments:
30-50bp homology target site-gal1promoter-30-50bp homology to insert
30-50bp homology to gal1 promoter-insert-30-50bp homology to target site
note that adding homology between gal and the insert will create total of 60bp-100bp homology without exceedingly long primers since the length is split between two primers (In my hands this length give you a high chance of success,however, the minimal homology of 30 can be used instead).
if you want to increase your chances of correct locus integration, you can select a counterselectable locus like can1 or ura3. Remember if you selection can be achieved from the gene that is under gal then you should recover in gal media before plating.
I have not studied this issue.
All best wishes for your continuing successes,
Prof.Dr.Otar Shainidze
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