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Problem with galactose induction

galactose induction

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#1 Orion



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Posted 25 January 2012 - 01:14 AM

Hi all,

I am having problems with getting expression of my genes which were cloned under the GAL1 promoter of pYES2/CT plasmid from invitrogen.

I had first inoculate the transformed yeast colonies in YNB -URA medium with 2% raffinose overnight. Then after around 12-16 hours, I transferred the cell pellets to the induction medium which is YNB -URA with 1% raffinose and 2% galactose. My cells were growing normally with OD600 peaked at a reading of around 13. I had collected samples from the induction medium from 19 hours to 71 hours after induction. Samples were taken every 12 hours after the 19th hour of induction. I tried to run western blot and even RT-PCR but all tests gave me negative results. My genes were successfully transformed inside the yeasts as confirmed by colony PCR. Thus, something must be wrong from the induction steps onwards.

I read it from somewhere that galactose induction occurs as early as 2 hours after induction. Is it true?
If true, then is it possible that by the 19th hour, all the galactose were used up and thus I am getting negative results?
Hope someone will be kind enough to offer some advices on this. I had be trying at this for nearly 2 months now. Any help will be greatly appreciated. Thank you in advance.


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