IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

[Yeast] Re: Yeast Digest, Vol 31, Issue 5

vipulkumar parmar via yeast%40net.bio.net (by vipulgreat from gmail.com)
Sat Dec 15 20:03:26 EST 2007


I second the Qiagen protcol which is user modified protocol and has
usually worked for me.

cheers

Vipul

On 12/15/07, yeast-request from oat.bio.indiana.edu
<yeast-request from oat.bio.indiana.edu> wrote:
> Send Yeast mailing list submissions to
>         yeast from net.bio.net
>
> To subscribe or unsubscribe via the World Wide Web, visit
>         http://www.bio.net/biomail/listinfo/yeast
> or, via email, send a message with subject or body 'help' to
>         yeast-request from net.bio.net
>
> You can reach the person managing the list at
>         yeast-owner from net.bio.net
>
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Yeast digest..."
>
>
> Today's Topics:
>
>    1. Re: Yeast Digest, Vol 31, Issue 4 (Lionel Brooks)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Fri, 14 Dec 2007 12:35:36 -0500
> From: Lionel Brooks <lbthrice from Dartmouth.EDU>
> Subject: [Yeast] Re: Yeast Digest, Vol 31, Issue 4
> To: yeast from oat.bio.indiana.edu
> Message-ID: <4762BEE8.9080108 from dartmouth.edu>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> yeast-request from oat.bio.indiana.edu wrote:
> > Send Yeast mailing list submissions to
> >       yeast from net.bio.net
> >
> > To subscribe or unsubscribe via the World Wide Web, visit
> >       http://www.bio.net/biomail/listinfo/yeast
> > or, via email, send a message with subject or body 'help' to
> >       yeast-request from net.bio.net
> >
> > You can reach the person managing the list at
> >       yeast-owner from net.bio.net
> >
> > When replying, please edit your Subject line so it is more specific
> > than "Re: Contents of Yeast digest..."
> >
> >
> > Today's Topics:
> >
> >    1. Extraction of Plasmid from S. cerevisiae. (lautys)
> >
> >
> > ----------------------------------------------------------------------
> >
> > 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
> > Message-ID:
> >       <c253195f0712122252i20184190haaf504d23b196e35 from mail.gmail.com>
> > Content-Type: text/plain; charset="iso-8859-1"
> >
> > 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
> > -------------- next part --------------
> > An HTML attachment was scrubbed...
> > URL: http://www.bio.net/bionet/mm/yeast/attachments/20071213/8920fa66/attachment-0001.html
> >
> > ------------------------------
> >
> > _______________________________________________
> > Yeast mailing list
> > Yeast from net.bio.net
> > http://www.bio.net/biomail/listinfo/yeast
> >
> > End of Yeast Digest, Vol 31, Issue 4
> > ************************************
> >
> 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
>
>
>
> ------------------------------
>
> _______________________________________________
> Yeast mailing list
> Yeast from net.bio.net
> http://www.bio.net/biomail/listinfo/yeast
>
> End of Yeast Digest, Vol 31, Issue 5
> ************************************
>



More information about the Yeast mailing list

Send comments to us at biosci-help [At] net.bio.net