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problem with yeast transformation


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#1 chocovac

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Posted 31 January 2010 - 12:08 PM

Hi all,

I had a URA marker-based plasmid (integrative version) transformed into S.cereviseae (BY strain). After 3 days, I see really tiny colonies (that appeared on 2nd day afternoon) that don't seem to grow at all. The control plate (no vector) is clean. Usually, after three days, the colonies get obviously big to be picked and plated again.

The colonies on the plate are so small (literally, tiny dots) that I am not sure if it is yeast. I cannot reason out that it could be contamination or am I wrong here?

Share your thoughts guys.


Thanks,
Choco

#2 saeid ziaei

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Posted 27 June 2010 - 09:46 AM

what is your media ? is it from good supplier ?

#3 perneseblue

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Posted 03 July 2010 - 08:54 PM

how is your plating density? If it is high, it is possible for yeast cells to turn "cannibal" and scavenge missing uracil from dead cells and support limited growth. I usually spend the extra few plates to keep the plating density low.

Do these microcolonies grow well once re-plated on a second selection plate?

Does the plasmid carry/express any genes? Expression of that gene could make the yeast cell very sick thus the micro colony.

So... I would pick these micro-colonies and streak them onto a second identical selection plate. If the streak grows, it is fine (probably). If the micro-colony does not grow on the second selection plate, experience indicates that isn't anything there.
May your PCR products be long, your protocols short and your boss on holiday




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