[Yeast] Re: Yeast Digest, Vol 31, Issue 4

Lionel Brooks via yeast%40net.bio.net (by lbthrice from Dartmouth.EDU)
Fri Dec 14 12:35:36 EST 2007


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> Today's Topics:
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>    1. Extraction of Plasmid from S. cerevisiae. (lautys)
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> Message: 1
> Date: Thu, 13 Dec 2007 14:52:45 +0800
> From: lautys <lautys from gmail.com>
> Subject: [Yeast] Extraction of Plasmid from S. cerevisiae.
> To: yeast from magpie.bio.indiana.edu
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> Hello,
>
> 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.
>
> Lau
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>   
Hi there,

Before saying this, I declare no competing financial interests =)
At any rate, Qiagen has posted a modified plasmid isolation protocol for 
their QIAprep Spin Miniprep kit.  It worked for me and it was quick and 
easy.  You can find it at the Qiagen site. 

Find it here
http://www1.qiagen.com/literature/handbooks/literature.aspx?id=1000248

I wonder if that is a low-copy plasmid?  If it is then expect 2 copies 
per cell.


-Lionel Brooks



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