From Arun.Thomas from Biologie.Uni-Osnabrueck.DE Wed Feb 4 05:36:10 2009 From: Arun.Thomas from Biologie.Uni-Osnabrueck.DE (Thomas, Arun) Date: Wed Feb 4 06:47:45 2009 Subject: [Yeast] raffinose metabolism Message-ID: Hi everyone, To induce a gene under the GAL promoter from a 2micron plasmid, why is it preferred to grow the cells in raffinose/glucose initially followed by induction with galactose? If cells grow slowly in galactose, why is it so? Thank you, Thomas From cartera from cmp.ucsf.edu Wed Feb 4 12:27:26 2009 From: cartera from cmp.ucsf.edu (Andrew Carter) Date: Wed Feb 4 12:50:45 2009 Subject: [Yeast] RE: Yeast Digest, Vol 45, Issue 1 In-Reply-To: <200902041706.n14H65806400@net.bio.net> References: <200902041706.n14H65806400@net.bio.net> Message-ID: <000001c986ed$daa298f0$8fe7cad0$@ucsf.edu> Hi Thomas Glucose represses the Gal promoter whereas Raffinose doesn't. If you try growing up yeast in YPD (2% glucose) and then add galactose you will get no (in my experience) induction. If you grow up in YP raffinose and add galactose the promoter will get turned on immediately. It turns out however that you can first grow in glucose and then add galactose. You just have to make sure that you have used up all your glucose. I have been playing with this quite a lot recently (in a large fermentor) and it works well. The key for me was using 2X YP and 1% glucose and waiting until growth plateaus. Then add in 2% galactose (0.5% and 1% also work) and induction turned on just fine. I was using induction from a gal promoter inserted in the genome and haven't tried it with a plasmid based system, but my guess is it should work. The real advantage is that Raffinose gets really expensive if you want to scale up... Not sure why growth is slower in galactose than glucose but I see it alot (final OD is usually lower as well). Best wishes Andrew -----Original Message----- From: yeast-bounces@oat.bio.indiana.edu [mailto:yeast-bounces@oat.bio.indiana.edu] On Behalf Of yeast-request@oat.bio.indiana.edu Sent: Wednesday, February 04, 2009 9:06 AM To: yeast@magpie.bio.indiana.edu Subject: Yeast Digest, Vol 45, Issue 1 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. raffinose metabolism (Thomas, Arun) ---------------------------------------------------------------------- Message: 1 Date: Wed, 4 Feb 2009 11:36:10 +0100 From: "Thomas, Arun" Subject: [Yeast] raffinose metabolism To: Message-ID: Content-Type: text/plain; charset="iso-8859-1" Hi everyone, To induce a gene under the GAL promoter from a 2micron plasmid, why is it preferred to grow the cells in raffinose/glucose initially followed by induction with galactose? If cells grow slowly in galactose, why is it so? Thank you, Thomas ------------------------------ _______________________________________________ Yeast mailing list Yeast@net.bio.net http://www.bio.net/biomail/listinfo/yeast End of Yeast Digest, Vol 45, Issue 1 ************************************ From d_bosch from hotmail.com Thu Feb 5 13:01:22 2009 From: d_bosch from hotmail.com (Daniel Bosch) Date: Thu Feb 5 15:06:06 2009 Subject: [Yeast] RE: Yeast Digest, Vol 45, Issue 1 In-Reply-To: <000001c986ed$daa298f0$8fe7cad0$@ucsf.edu> References: <200902041706.n14H65806400@net.bio.net> <000001c986ed$daa298f0$8fe7cad0$@ucsf.edu> Message-ID: Hi there, Baker's yeasts loves glucose and fructose, these are consumed earlier than any other carbon source present in the medium. Uptake and degradation of galactose (and other respiratory carbon sources) is not functional for as long as glucose is available. Therefore, to get the GAL promoter to work glucose mustn't be present. That's why yeast are grown on raffinose, but in principle you could used cheaper carbon sources (e.g. maltose, ethanol, acetate). Glucose fermentation yields more energy per unit of time than galactose or raffinose, hence glucose allows the fastest growth rate. In addition, during glucose growth a large portion of the genome is repressesed saving energy, therefore glucose yields a higher OD than other carbon sources. Hope this helps you, Daniel Bosch -------------------------------------------------- From: "Andrew Carter" Sent: Wednesday, February 04, 2009 6:27 PM To: ; Subject: [Yeast] RE: Yeast Digest, Vol 45, Issue 1 > Hi Thomas > Glucose represses the Gal promoter whereas Raffinose doesn't. If you try > growing up yeast in YPD (2% glucose) and then add galactose you will get > no > (in my experience) induction. If you grow up in YP raffinose and add > galactose the promoter will get turned on immediately. > > It turns out however that you can first grow in glucose and then add > galactose. You just have to make sure that you have used up all your > glucose. I have been playing with this quite a lot recently (in a large > fermentor) and it works well. The key for me was using 2X YP and 1% > glucose > and waiting until growth plateaus. Then add in 2% galactose (0.5% and 1% > also work) and induction turned on just fine. I was using induction from > a > gal promoter inserted in the genome and haven't tried it with a plasmid > based system, but my guess is it should work. The real advantage is that > Raffinose gets really expensive if you want to scale up... > > Not sure why growth is slower in galactose than glucose but I see it alot > (final OD is usually lower as well). > Best wishes > Andrew > > > > > > > -----Original Message----- > From: yeast-bounces@oat.bio.indiana.edu > [mailto:yeast-bounces@oat.bio.indiana.edu] On Behalf Of > yeast-request@oat.bio.indiana.edu > Sent: Wednesday, February 04, 2009 9:06 AM > To: yeast@magpie.bio.indiana.edu > Subject: Yeast Digest, Vol 45, Issue 1 > > 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. raffinose metabolism (Thomas, Arun) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 4 Feb 2009 11:36:10 +0100 > From: "Thomas, Arun" > Subject: [Yeast] raffinose metabolism > To: > Message-ID: > > > > Content-Type: text/plain; charset="iso-8859-1" > > Hi everyone, > > To induce a gene under the GAL promoter from a 2micron plasmid, why is it > preferred to grow the cells in raffinose/glucose initially followed by > induction with galactose? > > If cells grow slowly in galactose, why is it so? > > Thank you, > Thomas > > > > ------------------------------ > > _______________________________________________ > Yeast mailing list > Yeast@net.bio.net > http://www.bio.net/biomail/listinfo/yeast > > End of Yeast Digest, Vol 45, Issue 1 > ************************************ > > _______________________________________________ > Yeast mailing list > Yeast@net.bio.net > http://www.bio.net/biomail/listinfo/yeast > From S.Wang from uws.edu.au Thu Feb 5 14:09:39 2009 From: S.Wang from uws.edu.au (Shaoyu Wang) Date: Thu Feb 5 15:06:11 2009 Subject: [Yeast] RE: Yeast Digest, Vol 45, Issue 2 References: <200902051706.n15H6E823663@net.bio.net> Message-ID: Hello, Everyone, FCY2 gene is an important purine-cytosine permease. However, there two more putative purine-cytosine permeases encoded by FCY21 and FCY22. But there is lack of information on exact substrates and regulation of their activity etc. Only information I get is: Fcy21p and Fcy22p are very similar to Fcy2p but can not substitute its function. I wonder if anyone have more information on FCY21 and FCY22 and their protein product functions. Any pointers are appreciated. Thank you Shaun ________________________________ From: yeast-bounces@oat.bio.indiana.edu on behalf of yeast-request@oat.bio.indiana.edu Sent: Fri 6/02/2009 4:06 AM To: yeast@magpie.bio.indiana.edu Subject: Yeast Digest, Vol 45, Issue 2 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 45, Issue 1 (Andrew Carter) ---------------------------------------------------------------------- Message: 1 Date: Wed, 4 Feb 2009 09:27:26 -0800 From: "Andrew Carter" Subject: [Yeast] RE: Yeast Digest, Vol 45, Issue 1 To: , Message-ID: <000001c986ed$daa298f0$8fe7cad0$@ucsf.edu> Content-Type: text/plain; charset="us-ascii" Hi Thomas Glucose represses the Gal promoter whereas Raffinose doesn't. If you try growing up yeast in YPD (2% glucose) and then add galactose you will get no (in my experience) induction. If you grow up in YP raffinose and add galactose the promoter will get turned on immediately. It turns out however that you can first grow in glucose and then add galactose. You just have to make sure that you have used up all your glucose. I have been playing with this quite a lot recently (in a large fermentor) and it works well. The key for me was using 2X YP and 1% glucose and waiting until growth plateaus. Then add in 2% galactose (0.5% and 1% also work) and induction turned on just fine. I was using induction from a gal promoter inserted in the genome and haven't tried it with a plasmid based system, but my guess is it should work. The real advantage is that Raffinose gets really expensive if you want to scale up... Not sure why growth is slower in galactose than glucose but I see it alot (final OD is usually lower as well). Best wishes Andrew -----Original Message----- From: yeast-bounces@oat.bio.indiana.edu [mailto:yeast-bounces@oat.bio.indiana.edu] On Behalf Of yeast-request@oat.bio.indiana.edu Sent: Wednesday, February 04, 2009 9:06 AM To: yeast@magpie.bio.indiana.edu Subject: Yeast Digest, Vol 45, Issue 1 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. raffinose metabolism (Thomas, Arun) ---------------------------------------------------------------------- Message: 1 Date: Wed, 4 Feb 2009 11:36:10 +0100 From: "Thomas, Arun" Subject: [Yeast] raffinose metabolism To: Message-ID: Content-Type: text/plain; charset="iso-8859-1" Hi everyone, To induce a gene under the GAL promoter from a 2micron plasmid, why is it preferred to grow the cells in raffinose/glucose initially followed by induction with galactose? If cells grow slowly in galactose, why is it so? Thank you, Thomas ------------------------------ _______________________________________________ Yeast mailing list Yeast@net.bio.net http://www.bio.net/biomail/listinfo/yeast End of Yeast Digest, Vol 45, Issue 1 ************************************ ------------------------------ _______________________________________________ Yeast mailing list Yeast@net.bio.net http://www.bio.net/biomail/listinfo/yeast End of Yeast Digest, Vol 45, Issue 2 ************************************ -------------- next part -------------- A non-text attachment was scrubbed... Name: not available Type: application/ms-tnef Size: 8042 bytes Desc: not available Url : http://www.bio.net/bionet/mm/yeast/attachments/20090206/c44167cf/attachment.bin From cedric.desmet from gmail.com Fri Feb 6 12:12:41 2009 From: cedric.desmet from gmail.com (Cedric) Date: Sat Feb 7 10:51:11 2009 Subject: [Yeast] Re: Yeast Digest, Vol 45, Issue 3 In-Reply-To: <200902061705.n16H5g827279@net.bio.net> References: <200902061705.n16H5g827279@net.bio.net> Message-ID: <827989df0902060912n42f94390t6eedf385e0faeb2a@mail.gmail.com> Hi all, A related question: are raffinose and galactose fermentable carbon sources? I read somewhere that they are "poorly fermentable" and when looking at the lipid profiles, I suspect that they are not fermenting. Is this true, cause in theory they are sugars and should be fermentable. Kind regards, Cedric On Fri, Feb 6, 2009 at 6:05 PM, 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: RE: Yeast Digest, Vol 45, Issue 1 (Daniel Bosch) > 2. RE: Yeast Digest, Vol 45, Issue 2 (Shaoyu Wang) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Thu, 5 Feb 2009 19:01:22 +0100 > From: "Daniel Bosch" > Subject: Re: [Yeast] RE: Yeast Digest, Vol 45, Issue 1 > To: , > Message-ID: > Content-Type: text/plain; format=flowed; charset="iso-8859-1"; > reply-type=original > > Hi there, > > Baker's yeasts loves glucose and fructose, these are consumed earlier than > any other carbon source present in the medium. Uptake and degradation of > galactose (and other respiratory carbon sources) is not functional for as > long as glucose is available. Therefore, to get the GAL promoter to work > glucose mustn't be present. That's why yeast are grown on raffinose, but in > principle you could used cheaper carbon sources (e.g. maltose, ethanol, > acetate). Glucose fermentation yields more energy per unit of time than > galactose or raffinose, hence glucose allows the fastest growth rate. In > addition, during glucose growth a large portion of the genome is > repressesed > saving energy, therefore glucose yields a higher OD than other carbon > sources. > > Hope this helps you, > > Daniel Bosch > -------------------------------------------------- > From: "Andrew Carter" > Sent: Wednesday, February 04, 2009 6:27 PM > To: ; > Subject: [Yeast] RE: Yeast Digest, Vol 45, Issue 1 > > > Hi Thomas > > Glucose represses the Gal promoter whereas Raffinose doesn't. If you try > > growing up yeast in YPD (2% glucose) and then add galactose you will get > > no > > (in my experience) induction. If you grow up in YP raffinose and add > > galactose the promoter will get turned on immediately. > > > > It turns out however that you can first grow in glucose and then add > > galactose. You just have to make sure that you have used up all your > > glucose. I have been playing with this quite a lot recently (in a large > > fermentor) and it works well. The key for me was using 2X YP and 1% > > glucose > > and waiting until growth plateaus. Then add in 2% galactose (0.5% and 1% > > also work) and induction turned on just fine. I was using induction from > > a > > gal promoter inserted in the genome and haven't tried it with a plasmid > > based system, but my guess is it should work. The real advantage is that > > Raffinose gets really expensive if you want to scale up... > > > > Not sure why growth is slower in galactose than glucose but I see it alot > > (final OD is usually lower as well). > > Best wishes > > Andrew > > > > > > > > > > > > > > -----Original Message----- > > From: yeast-bounces@oat.bio.indiana.edu > > [mailto:yeast-bounces@oat.bio.indiana.edu] On Behalf Of > > yeast-request@oat.bio.indiana.edu > > Sent: Wednesday, February 04, 2009 9:06 AM > > To: yeast@magpie.bio.indiana.edu > > Subject: Yeast Digest, Vol 45, Issue 1 > > > > 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. raffinose metabolism (Thomas, Arun) > > > > > > ---------------------------------------------------------------------- > > > > Message: 1 > > Date: Wed, 4 Feb 2009 11:36:10 +0100 > > From: "Thomas, Arun" > > Subject: [Yeast] raffinose metabolism > > To: > > Message-ID: > > > > > > > > Content-Type: text/plain; charset="iso-8859-1" > > > > Hi everyone, > > > > To induce a gene under the GAL promoter from a 2micron plasmid, why is it > > preferred to grow the cells in raffinose/glucose initially followed by > > induction with galactose? > > > > If cells grow slowly in galactose, why is it so? > > > > Thank you, > > Thomas > > > > > > > > ------------------------------ > > > > _______________________________________________ > > Yeast mailing list > > Yeast@net.bio.net > > http://www.bio.net/biomail/listinfo/yeast > > > > End of Yeast Digest, Vol 45, Issue 1 > > ************************************ > > > > _______________________________________________ > > Yeast mailing list > > Yeast@net.bio.net > > http://www.bio.net/biomail/listinfo/yeast > > > > > > ------------------------------ > > Message: 2 > Date: Fri, 6 Feb 2009 06:09:39 +1100 > From: "Shaoyu Wang" > Subject: [Yeast] RE: Yeast Digest, Vol 45, Issue 2 > To: > Message-ID: > > Content-Type: text/plain; charset="iso-8859-1" > > Hello, Everyone, > > FCY2 gene is an important purine-cytosine permease. However, there two more > putative purine-cytosine permeases encoded by FCY21 and FCY22. But there is > lack of information on exact substrates and regulation of their activity > etc. Only information I get is: Fcy21p and Fcy22p are very similar to Fcy2p > but can not substitute its function. > > I wonder if anyone have more information on FCY21 and FCY22 and their > protein product functions. Any pointers are appreciated. > > Thank you > > Shaun > > ________________________________ > > From: yeast-bounces@oat.bio.indiana.edu on behalf of > yeast-request@oat.bio.indiana.edu > Sent: Fri 6/02/2009 4:06 AM > To: yeast@magpie.bio.indiana.edu > Subject: Yeast Digest, Vol 45, Issue 2 > > > > 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 45, Issue 1 (Andrew Carter) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 4 Feb 2009 09:27:26 -0800 > From: "Andrew Carter" > Subject: [Yeast] RE: Yeast Digest, Vol 45, Issue 1 > To: , > Message-ID: <000001c986ed$daa298f0$8fe7cad0$@ucsf.edu> > Content-Type: text/plain; charset="us-ascii" > > Hi Thomas > Glucose represses the Gal promoter whereas Raffinose doesn't. If you try > growing up yeast in YPD (2% glucose) and then add galactose you will get no > (in my experience) induction. If you grow up in YP raffinose and add > galactose the promoter will get turned on immediately. > > It turns out however that you can first grow in glucose and then add > galactose. You just have to make sure that you have used up all your > glucose. I have been playing with this quite a lot recently (in a large > fermentor) and it works well. The key for me was using 2X YP and 1% > glucose > and waiting until growth plateaus. Then add in 2% galactose (0.5% and 1% > also work) and induction turned on just fine. I was using induction from a > gal promoter inserted in the genome and haven't tried it with a plasmid > based system, but my guess is it should work. The real advantage is that > Raffinose gets really expensive if you want to scale up... > > Not sure why growth is slower in galactose than glucose but I see it alot > (final OD is usually lower as well). > Best wishes > Andrew > > > > > > > -----Original Message----- > From: yeast-bounces@oat.bio.indiana.edu > [mailto:yeast-bounces@oat.bio.indiana.edu] On Behalf Of > yeast-request@oat.bio.indiana.edu > Sent: Wednesday, February 04, 2009 9:06 AM > To: yeast@magpie.bio.indiana.edu > Subject: Yeast Digest, Vol 45, Issue 1 > > 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. raffinose metabolism (Thomas, Arun) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 4 Feb 2009 11:36:10 +0100 > From: "Thomas, Arun" > Subject: [Yeast] raffinose metabolism > To: > Message-ID: > > > > Content-Type: text/plain; charset="iso-8859-1" > > Hi everyone, > > To induce a gene under the GAL promoter from a 2micron plasmid, why is it > preferred to grow the cells in raffinose/glucose initially followed by > induction with galactose? > > If cells grow slowly in galactose, why is it so? > > Thank you, > Thomas > > > > ------------------------------ > > _______________________________________________ > Yeast mailing list > Yeast@net.bio.net > http://www.bio.net/biomail/listinfo/yeast > > End of Yeast Digest, Vol 45, Issue 1 > ************************************ > > > > ------------------------------ > > _______________________________________________ > Yeast mailing list > Yeast@net.bio.net > http://www.bio.net/biomail/listinfo/yeast > > End of Yeast Digest, Vol 45, Issue 2 > ************************************ > > > -------------- next part -------------- > A non-text attachment was scrubbed... > Name: not available > Type: application/ms-tnef > Size: 8042 bytes > Desc: not available > Url : > http://www.bio.net/bionet/mm/yeast/attachments/20090206/c44167cf/attachment-0001.bin > > ------------------------------ > > _______________________________________________ > Yeast mailing list > Yeast@net.bio.net > http://www.bio.net/biomail/listinfo/yeast > > End of Yeast Digest, Vol 45, Issue 3 > ************************************ > -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/yeast/attachments/20090206/e3bfc5f5/attachment-0001.html From ranjuna.weerasekera from marquette.edu Sat Feb 7 14:20:55 2009 From: ranjuna.weerasekera from marquette.edu (Weerasekera, Ranjuna) Date: Sun Feb 8 13:32:12 2009 Subject: [Yeast] Re: Yeast Digest, Vol 45, Issue 3 In-Reply-To: <827989df0902060912n42f94390t6eedf385e0faeb2a@mail.gmail.com> References: <200902061705.n16H5g827279@net.bio.net>, <827989df0902060912n42f94390t6eedf385e0faeb2a@mail.gmail.com> Message-ID: <279388A6F536644CBBA99FB2263996CE6FDBD3661A@EMARQSTU1.marqnet.mu.edu> In a yeast speheroplast experiment combined with a Western Blot. How can you show that the yeast has lost it's cell wall? Thanks Ranjuna From clandry from post.harvard.edu Wed Feb 11 20:40:01 2009 From: clandry from post.harvard.edu (Christian Landry) Date: Wed Feb 11 23:40:46 2009 Subject: [Yeast] Multiple-Pcl deletion strain Message-ID: Hi, I am looking for a strain of S. cerevisae that would have all of its 10 Pcl (Pho85 cyclins: Pcl1, Pcl2, Pcl5, Pho80, Pcl6, Pcl7, Pcl8, Pcl9, Pcl10, Clg1) genes deleted. Thanks for any indication on where I could find one. Christian Landry clandry@post.harvard.edu From ZZhang from uwyo.edu Tue Feb 17 18:18:59 2009 From: ZZhang from uwyo.edu (Z.J. Zhang) Date: Wed Feb 18 09:52:06 2009 Subject: [Yeast] Mad2-GFP In-Reply-To: References: Message-ID: Hi all: Does anyone have experience with the Mad2-GFP strain from Invitrogen? I could not observe any GFP signal with it. I wonder if anything I did wrong, or something wrong the strain itself. Thank you for your help Zhaojie Zhang University of Wyoming From Jennifer.Chang from nyumc.org Thu Feb 19 17:32:26 2009 From: Jennifer.Chang from nyumc.org (Chang, Jennifer) Date: Thu Feb 19 17:49:10 2009 Subject: [Yeast] GAL4-DBD antibodies for immunofluorescence Message-ID: <628D3F39A80B5F47A4F231911D2B58CFCAEEB5@MSGWSDCPMB02.nyumc.org> Hello, All! I am looking for antibodies against the GAL4-DBD to verify that my bait constructs are indeed nuclear-localized. My bait is in pGBKT7 (Clontech Matchmaker). None of the commercially available antibodies (Clontech, Abcam, Santa Cruz) for GAL4-DBD are verified for immunofluorescence. I was wondering if anyone has used a GAL4-DBD antibody for IF and how well it worked. I have several bait constructs so I am hoping that I can use one antibody to verify nuclear localization for all of them. I only have an antibody against one of my baits and it works weakly for IF. Also, I would also welcome any comments, hints, suggestions, or advice on general yeast two-hybrid techniques (especially library transformation) prior to running my actual Y2H screen. Many thanks in advance! -Jennifer P.S. I can be e-mailed at jennifer.chang@nyumc.org or at jennifer.chang02@gmail.com ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= From aelkins from bostonproductions.com Fri Feb 20 09:56:47 2009 From: aelkins from bostonproductions.com (Arlene Elkins) Date: Fri Feb 20 12:09:30 2009 Subject: [Yeast] Need help for museum exhibit Message-ID: Could you please post the following to your members? I am working on an audio-visual exhibit for the Connecticut Science Center about bio-ethanol. I want to use footage of yeast budding to help illustrate the fermentation process. I would appreciate it if you have such footage, to contact me at aelkins@bostonproductions.com. I am happy to explain the project in greater detail and answer any questions. Thank you, Arlene Elkins Associate Producer Boston Productions BOSTON PRODUCTIONS INC. | imagine. what we do. ARLENE ELKINS | ASSOCIATE PRODUCER aelkins@bostonproductions.com | p 781.255.1555 x219 | f 781.255.1556 -------------- next part -------------- An HTML attachment was scrubbed... URL: http://www.bio.net/bionet/mm/yeast/attachments/20090220/e56a47eb/attachment.html From birky from u.arizona.edu Fri Feb 20 21:52:25 2009 From: birky from u.arizona.edu (Bill Birky) Date: Sun Feb 22 10:16:42 2009 Subject: [Yeast] Cytoduction Message-ID: Chromosomes are transferred between nuclei in KAR/kar heterokaryons. Does anybody know the physical mechanism of the transfer, and why it seems to be limited to one chromosome at a time? Thanks, Bill Birky -- C. William Birky, Jr. Professor Emeritus Department of Ecology and Evolutionary Biology University of Arizona Tucson, AZ 85721 From ranjuna.weerasekera from marquette.edu Wed Feb 25 14:13:49 2009 From: ranjuna.weerasekera from marquette.edu (Weerasekera, Ranjuna) Date: Thu Feb 26 13:43:22 2009 Subject: [Yeast] Looking for an explanation Message-ID: <279388A6F536644CBBA99FB2263996CE7011FA3165@EMARQSTU1.marqnet.mu.edu> I'm expressing a plant cold responsive protein in yeast to study if the protein protects yeast during cold conditions. The protein is predicted to be about 10kDa. But on my Western it is detected at about 15-21kDa, with multiple bands. Dose anyone have any good explanations for this phenomenon? Thank you RW