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[Yeast] Extraction of Plasmid from S. cerevisiae.

lautys via yeast%40net.bio.net (by lautys from gmail.com)
Thu Dec 13 01:52:45 EST 2007


I am doing yeast (*S. cerevisiae*) transformation for the first time.

My current work flow is:

ligation of yeast plasmid (pYC2/NT A) and gene of interest --> "1st" *E.
coli *transformation --> plasmid extraction (check with enzyme digestion)
--> yeast (INV*Sc*1) transformation --> plasmid extraction --> "2nd" *E.
coli *transformation --> plasmid extraction (check with enzyme digestion)
--> yeast (INV*Sc*1) transformation --> over-expression of the protein

I have tried few method including Gietz, R.D. & Schiestl, R.H. *Quick and
Easy Yeast Transformation Using the LiAc/SS Carrier DNA/PEG Method*. *Natural
Protocols* (2007), and find the transformation yields are very satisfying.

But I am facing problem in plasmid extraction from yeast where I am getting
low *E. coli *transformants. The yields are low in term of lower in number
of colonies and smaller colonies, compare to 1st *E. coli *transformation.

For rapid isolation of plasmid from yeast, I am currently using both
original and lab protocol which modified from Ausubel F.M., Brent, R.,
Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., & Struhl,
K. *Short
protocols in molecular biology: a compendium of methods from current
protocols in molecular biology (5th ed)*, *New York: John Wiley & Sons, Inc*.
(2002), and Robzyk, K., and Kassir, Yona., *A simple and highly efficient
procedure for rescuing autonomous plasmids from yeast*, *Nucleic Acids
Research* (1992).

I would like to ask:

   1. is it normal for the "2nd" transformation having lower yield
   compare to the "1st"?
   2. if not, any other suggestion of plasmid extraction? i am using
   beads in the extraction.
   3. is the "2nd" *E. coli *transformation necessary? this step was
   include because most of the protocol i read do so, but i do not understand
   why. Can somebody brief explain? or suggest a reading material?

Thank you very much.

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