From mccus001 from mc.duke.edu Mon Dec 3 11:23:37 2007 From: mccus001 from mc.duke.edu (John McCusker) Date: Mon Dec 3 14:58:44 2007 Subject: [Yeast] Mating type switching in S. cerevisiae Katrin Hartwich Message-ID: Hello, My former advisor, Jim Haber, provided SGD with some text that explains the problem: see http://db.yeastgenome.org/cgi-bin/locus.pl?locus=mat then go to "summary paragraph" http://db.yeastgenome.org/cgi-bin/locus.pl?locus=mat#summaryParagraph Basically, MATa in the S288c background (BY4141 is S288c background) has a single base pair polymorphism that greatly impairs (but does not completely prevent) MAT switching. Regards, John McCusker ***************************************** Hello to all, I'm working with the S. cerevisiae strains from the EUROSCARF collection (BY4741). All strains have the mating type a and I want to switch now the mating type of some of them to alpha. I have a protocol and a plasmid for mating type switching from a friend but it didn't work that well. Does anybody have a good working protocol/plasmid for mating type switching for me? Thank's a lot! Best wishes, Katrin -- Katrin Hartwich European Neuroscience Institute G?ttingen (ENI-G) Molecular Neurogenetics Grisebachstra?e 5 37073 G?ttingen Germany Phone: +49 (0)551-39-13972 Fax: +49 (0)551-39-12346 Email: khartwi from gwdg.de -- ************************************************* Note: The material reflected in this message/document is considered sensitive and should be distributed only on a Need-to-Know basis. This message content/document is for the exclusive use of the intended recipient, should be considered confidential, and is considered the sole property of Duke University. If you believe that you have been sent this message in error, please notify the sender. John H. McCusker, Assoc. Prof. Dept. of Molecular Genetics & Microbiology, 3020 Duke University Medical Center Durham, NC 27710 http://www.duke.edu/web/microlabs/mccusker/ phone: (919) 681-6744 fax: (919) 684-8735 e-mail: mccus001@mc.duke.edu PLASMID REQUESTS: PLEASE CHECK WEB SITE -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/yeast/attachments/20071203/061eb0de/attachment.html From clandry from post.harvard.edu Mon Dec 10 11:09:21 2007 From: clandry from post.harvard.edu (Christian Landry) Date: Tue Dec 11 09:32:00 2007 Subject: [Yeast] Sacc. Kluyveri Message-ID: <907321F6-D42D-4370-9D04-152DDE50338C@post.harvard.edu> Hello, Does anyone know if it is feasible to do homologous recombination (with PCR products) of Saccharomyces kluyveri? Do you know any reference? Finally, do you know who would have haploid strains (a and alpha) for this species with selectable markers? Thanks a lot, Christian -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/yeast/attachments/20071210/83bcf43d/attachment.html From pmmagic from gmail.com Tue Dec 11 15:04:33 2007 From: pmmagic from gmail.com (paul m) Date: Tue Dec 11 15:16:46 2007 Subject: [Yeast] Sacc. Kluyveri In-Reply-To: <907321F6-D42D-4370-9D04-152DDE50338C@post.harvard.edu> References: <907321F6-D42D-4370-9D04-152DDE50338C@post.harvard.edu> Message-ID: <991e7bc10712111204w3ad9b34bqe3119e83ba3a8f0d@mail.gmail.com> A book that might be useful is: Wolf, Bruenig, Barth (Eds.). 2003. Non-convential yeasts in genetics, biochemistry, and biotechnology. Springer. There's nothing specifically on S. kluyveri but it does have a couple of protocols for doing gene disruptions in K. lactis which is known to exhibit low rates of homologous recombination. Cheers, Paul Magwene On Dec 10, 2007 11:09 AM, Christian Landry wrote: > Hello, > Does anyone know if it is feasible to do homologous recombination (with > PCR products) of Saccharomyces kluyveri? Do you know any reference? > Finally, do you know who would have haploid strains (a and alpha) for > this species with selectable markers? > > Thanks a lot, > > Christian > > > > _______________________________________________ > Yeast mailing list > Yeast@net.bio.net > http://www.bio.net/biomail/listinfo/yeast > -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/yeast/attachments/20071211/5341af50/attachment.html From lautys from gmail.com Thu Dec 13 01:52:45 2007 From: lautys from gmail.com (lautys) Date: Fri Dec 14 00:24:47 2007 Subject: [Yeast] Extraction of Plasmid from S. cerevisiae. Message-ID: 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.html From lbthrice from Dartmouth.EDU Fri Dec 14 12:35:36 2007 From: lbthrice from Dartmouth.EDU (Lionel Brooks) Date: Fri Dec 14 16:01:09 2007 Subject: [Yeast] Re: Yeast Digest, Vol 31, Issue 4 In-Reply-To: <200712141705.lBEH5xY22205@net.bio.net> References: <200712141705.lBEH5xY22205@net.bio.net> Message-ID: <4762BEE8.9080108@dartmouth.edu> yeast-request@oat.bio.indiana.edu wrote: > Send Yeast mailing list submissions to > yeast@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@net.bio.net > > You can reach the person managing the list at > yeast-owner@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 > Subject: [Yeast] Extraction of Plasmid from S. cerevisiae. > To: yeast@magpie.bio.indiana.edu > Message-ID: > > 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@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 From vipulgreat from gmail.com Sat Dec 15 20:03:26 2007 From: vipulgreat from gmail.com (vipulkumar parmar) Date: Sat Dec 15 22:22:19 2007 Subject: [Yeast] Re: Yeast Digest, Vol 31, Issue 5 In-Reply-To: <200712151704.lBFH4vY21583@net.bio.net> References: <200712151704.lBFH4vY21583@net.bio.net> Message-ID: <175e564a0712151703s60d70272lbd8eadf2559f128b@mail.gmail.com> I second the Qiagen protcol which is user modified protocol and has usually worked for me. cheers Vipul On 12/15/07, yeast-request@oat.bio.indiana.edu wrote: > Send Yeast mailing list submissions to > yeast@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@net.bio.net > > You can reach the person managing the list at > yeast-owner@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 > Subject: [Yeast] Re: Yeast Digest, Vol 31, Issue 4 > To: yeast@oat.bio.indiana.edu > Message-ID: <4762BEE8.9080108@dartmouth.edu> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed > > yeast-request@oat.bio.indiana.edu wrote: > > Send Yeast mailing list submissions to > > yeast@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@net.bio.net > > > > You can reach the person managing the list at > > yeast-owner@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 > > Subject: [Yeast] Extraction of Plasmid from S. cerevisiae. > > To: yeast@magpie.bio.indiana.edu > > Message-ID: > > > > 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@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@net.bio.net > http://www.bio.net/biomail/listinfo/yeast > > End of Yeast Digest, Vol 31, Issue 5 > ************************************ > From hannah.silver from jefferson.edu Fri Dec 21 17:08:51 2007 From: hannah.silver from jefferson.edu (Hannah R Silver) Date: Mon Dec 24 14:35:42 2007 Subject: [Yeast] alpha factor arresting in bar1 strains Message-ID: <40888dc80712211408t3a8210benc7c8ea962c21531b@mail.gmail.com> alpha factor arresting in bar1 strains is a fairly common practice so i think that others must see this effect also. the first time i did the arrest the cells did not release synchronously and also did not release until 45-60min after washout (at 60min many cells were small budded but others we still shmooing). the next time i used a protease, pepsin, i washed 1x in YPR, 2x pepsin in YPR, 1x in YPR then released into YPR. still the cells did not release until after 45-60min but at least this time they were more synchronous but not quite perfect. ive ordered the pronase E protease that the koshland webpage recommends but im not sure if the delayed in the release of the cells from G1 is common for others. can anyone advise here? **************************************** HR Silver PhD Candidate Department of Biochemistry and Molecular Biology Thomas Jefferson University 233 South 10th Street, BLSB 231 Philadelphia, Pennsylvania 19107