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yeast transformation


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#1 Penny

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Posted 27 May 2011 - 08:37 PM

I had one problem for yeast transformation. I followed the LiAc/PEG yeast transformation protocol. The same gene was transformed twice with the same protocol. For the first transformation, the yeast cells only took 40h induction to reach OD value of 8. But for the second set, the yeast cells grew really slowly. The only different for these two transformations I can think of is the freshness of the competent cell. I used the freshly prepared yeast competent cell for the first transformation. For second time, I used the left competent cells made from the first time (about one week later). The cells were kept in -80 degree in 1ml of sterial water. So, my question is do you have to prepare the fresh yeast competent cells every time? And what could be other reasons for the different yeast growth rate.

#2 kajmak

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Posted 28 May 2011 - 11:14 PM

It is better to prepare competent cells every time you need them. I never used cell older than 24 h, and kept them at 4 C. Lots of cells die after taking them from -80. When you make cells any OD from 0.4 to 0.8 is good (lower the better) so you do not need to dedicate whole day to yeast.
Transformed yeast are behaving different every time. You have to keep an eye on incubation conditions, media volume, size of a flask or whatever you induce them in, everything has to be the same every time and even then there are variations. The thing with yeast is, not all of them are transformed, I mean, they lose introduced plasmid relatively fast and they start behaving differently.




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