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Why must I go through an additional overnight growth step in SD-ura raffinose media if I already grew and froze down stocks of the yeast ORF strain in SD-ura?
You cannot simply take a 1ml frozen stock in SD-ura 8% glycerol, add them to 1 L of YP galactose and grow overnight. The ORF clones are under a GAL promoter. If one has any glucose in the media or the clone has not shifted over to the use of galactose, then you will not see any protein expression. The overnight in raffinose allows for all the glucose & glucose metabolism products to clear before switching to galactose. It will not work to go straight to galactose (we've tried it several times).