Hello everyone,
I am doing yeast transformation? I should grow the starting yeast in 5-FoA/YPD to select URA(-) strains. Why do I need to do this step? Is it because URA(+) strains habouring URA gene which is a reporter gene will interfere the transformation of my gene of interest? What will happen if I transfer my target gene into a URA(+) strain?
Thank you very much.
Dear Van Phung
You need to screen the URA(-) strains prior to transformation to confirm the uracil auxotrophs. 5-FoA is derivative of a pyrimidine precursor orotic acid. If orotidine 5-monophosphate decarboxylase gene is present, i.e. URA(+), it can enzymatically convert 5-fluoroorotic acid into a toxic metabolite. Therefore, you need this step before transformation.
For your transformation, you must be using URA3 gene as the auxotrophic marker. So, you need to select URA(-) strains for the transformation and seed the transformants onto uracil free selective plate. If you use URA(+) strain for the transformation, you cannot select the positive transformants, because all the strains carrying URA3 gene will grow onto the selective plate, therefore, you cannot isolate the transformants which are truly transformed.
Best wishes.
Satyendra
Hi there,
You need a Ura- strain because the plasmid you will use for transformation bears URA3 gene which will allow the transformant to become Ura+. Plating the transformation mix on SCM lacking uracil will select the cells with plasmid.
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