I am working with bog-standard yeast and have to transfer my first culture into an induction medium. The manufacturers protocol for the used vector says to perform this centrifugation and washing step at 4°C, but I'd like to hear your opinions why I am supposed to expose my culture to this temperature shock of 24 K twice.
When harvesting and lysing the yeast I see the purpose of cooling down, but when simply transferring it to another medium I'd expect a longer lag phase when compared to centrifugation at about 28°C.
I'd like to hear your opinion on this procedure. Is there some sense behind it or is it simply 4°C because most stuff has to be centrifuged at this temperature anyways?
Hi there,
There is no point working at low temperature : time needed for the full process (harvesting, resuspension into wash solution, harvesting again) is pretty short . I would also suggest to prewarm the induction media in order to shorten the lag phase.
Hi Andre,
Well! Everything depends on the type of induction you are using. I'd liek to learn a bit more about that. Some induction can be carried out WITHOUT any centrifugation and washings steps! Dominique was right in what he wrote!
Philippe
My yeast starting culture grows on glucose as carbon source, induction takes place in the presence of galactose. The ura3 deficient yeast was transformed with the high copy pYES2 vector from Invitrogen. The induction is inhibited by the presence of glucose, as far as we know, that's why we have to transfer the culture.
OK so temperature is definitely not a problem. The alternative to swtching carbone source for induction is to first grow cells in raffinose and then add galactose directly to the culture. Growth with raffinose is slower but induction with galactose is faster as raffinose has no inhibitory effect on gal promoters (unlike glucose).
I used to change the temperature from 4 degree to 25 degree or room temperature. I found that the cells did not pack well in warm temperature at the same speed of cold temp but the activity of both were the same.
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