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yeast glycerol stock


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#1 Penny

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Posted 27 May 2011 - 08:52 PM

I have already tranformed my gene of interest (encoding for certain enzymes) into the yeast cell. The yeast was induced for detecting enzyme activity. The high enzyme activity was observed. But I forgot to prepare the glycerol stock for the yeast cells. When I tried to repeat the experiment, I could not get the same enzyme acitivity at the this time. The same plasmid was used for transformation. I could not figure out why this is happennig. I still have some dried induced yeast cells in -80 from the first time. Can I prepare the glycerol stock from the induced yeast cells and then plate the glycerol stock and reinduced them again? Or is that any method I can use to extract the plasmid from these induced yeast cells to check the sequence of the gene?
Thanks,

Penny

#2 perneseblue

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Posted 28 May 2011 - 07:40 AM

If the plasmid has an origin of replication and a prokaryote selection marker it should be able to grow in E coli.

You can then extract DNA from the yeast and transform the DNA back into competent E coli.

When the expression experiment was repeated, was the growth conditions (culture media, culture size, temperature, shaking speed-oxygenation) changed? It could be that expression of the protein of interest is sensitive to these conditions.

As for re inducing yeast cells, I can not say.
May your PCR products be long, your protocols short and your boss on holiday

#3 Penny

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Posted 29 May 2011 - 07:26 AM

Thanks very much for the reply. I will try to extract the DNA out of yeast and sequence it first.




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