Nabble - Bio.net - Yeast

Re: Yeast Digest, Vol 54, Issue 3

2009-11-26T10:55:18Z

Hi ;
Use sporulation media for your mutant strain. this is the easy way I think.
on sporulation media if there is a spores that means your strain is somehow
diploid. You can check under microscope.

Alaattin KAYA

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Subject: Yeast Digest, Vol 54, Issue 3

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> 1. Phloxine B staining: diploidy or mutant phenotype?
> (Febe van Maldegem)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Wed, 25 Nov 2009 18:53:00 +0000
> From: Febe van Maldegem <maldegem@...>
> Subject: [Yeast] Phloxine B staining: diploidy or mutant phenotype?
> To: yeast@...
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> Dear colleagues,
>
> I have a temperature sensitive mutant strain, that also grows in red
> colonies on Phloxine B plates at 30C.
> How do I distinguish diploidy from a reduced viability due to the
> mutation? In other words, how can I reassure myself that I have a
> haploid mutant strain?
>
> Thanks in advance!
> Febe van Maldegem
> MRC-LMB, Cambridge, UK
>
>
>
> ------------------------------
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Phloxine B staining: diploidy or mutant phenotype?

2009-11-25T10:53:00Z

Dear colleagues,

I have a temperature sensitive mutant strain, that also grows in red
colonies on Phloxine B plates at 30C.
How do I distinguish diploidy from a reduced viability due to the
mutation? In other words, how can I reassure myself that I have a
haploid mutant strain?

Thanks in advance!
Febe van Maldegem
MRC-LMB, Cambridge, UK

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Slightly off topic: are home-baking and work-cell-culture incompatible?

2009-11-20T10:20:21Z

Hi everyone,

I've got a bit of a quandary and I'd like to hear your input. My
roommate bakes bread, and I do cell culture at the lab. I know that
some labs have experienced yeast contamination of their cell lines,
attributed to baking or brewing at home.

Do you have any particular tips, other than stringent good lab
practice, that can really help prevent any contamination? I can't
prohibit her from baking (nor do I want to), but are there any special
precautions that she/we can take at home?

And from your experience: are there any of you who do bake bread at
home, without problems at work? (am I worrying overly much?)

Thanks in advance,
Megan Barker

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Query Regarding Plasmid isolation protocol

2009-11-09T03:42:29Z

Does anybody preferably know the plasmid isolation from fission yeast?

Mukesh Pratap Yadav

Graduate student

C/O Prof. D.D. Dubey
Molecular Biology Laboratory
Department of Biotechnolgy

VBS Purvanchal University
Jaunpur-222001 (Uttar pradesh)
India.

Mob. No- +91-9451157085

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About Yeast

2009-10-30T03:45:26Z

Dear,

I am a Chemical Engineer. Please let me know the value you are mentioning
for yeast can that one be used for bioreactor modelling?

Thanks and Regards,
Sagar Lunawat

*********************************************************************************************************
I am a chemical engineer and am working on a project for the Yeast
Drying Plant at Coors.  My project requires that I know the density of
completely dry brewer's yeast.  

	So far, I have tried measuring the density in two ways (The yeast
contained 3% moisture).  First, I tried displacing the yeast in a known
amount of water to eliminate air space and came up with 344 lbm/bbl or
about 1.33 g/ml.  Then I tried packing the dry yeast and came up w
 ith
166 lbm/bbl or 0.6 g/ml.  I imagine the second way still contained a lot
of air and the density is greater than the second value.

	Please let me know what you think.

	Thanks!

Dear yeast! Get Yourself a cool, short @in.com Email ID now!

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Finding the TF for a short DNA regulatory sequences

2009-09-20T21:06:07Z

Dear There,

May I ask a simple help. I have used the RSAT - oligo-analysis tool and
obtained an enriched potential TF binding sequence such as "CCCCTC" in
the upstream (800) of my list of genes, how would I find the potential
TFs for this sequence. Any tools for this?

Thank you in advance

Shaun

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Subject: Yeast Digest, Vol 52, Issue 1

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Today's Topics:

1. transformation efficiencies in yeast (Francis Smet)

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Message: 1
Date: Wed, 2 Sep 2009 16:37:50 +0200
From: Francis Smet <francis.smet@...>
Subject: [Yeast] transformation efficiencies in yeast
To: yeast@...
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<2b5170340909020737y25741d4dnffb8b19c5dcca5a9@...>
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Dear all,

I'm wondering if there is a particular reason why invitrogen stopped
selling the pYD1 yeast surface display vector. An additional query
regarding this matter is if there is a link between the used selection
marker (trp in this case of pYD1) and library transformation
efficiencies. I noticed that newer YSD vectors do not use this selection
marker any more.

many thanks for your comments,

Cheers.
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S. bayanus strains

2009-09-14T12:09:48Z

Dear yeast researchers,

We are doing a pilot project looking at the polymorphism of some genes
in Saccharomyces bayanus. We would therefore need a sample of diverse
strains, either isolated recently from the wild or that have been in
the lab for a while but that were isolated from strains different from
the type strain that was sequenced.

Any help would greatly be appreciated,

Christian Landry

Laval University, Canada

clandry@...

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transformation efficiencies in yeast

2009-09-02T07:37:50Z

Dear all,

I'm wondering if there is a particular reason why invitrogen stopped selling the pYD1 yeast surface display vector. An additional query regarding this matter is if there is a link between the used selection marker (trp in this case of pYD1) and library transformation efficiencies. I noticed that newer YSD vectors do not use this selection marker any more.

many thanks for your comments,

Cheers.

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No background with yeast two-hybrid screen

2009-08-29T08:02:19Z

Hi Jennifer,

I have just recently performed a Y2HG screen and found that the plating volume of 200microlitres resulted in plates that were too dense – so much so, it looked like a lawn and individual colonies could not been seen at all.

I simply patched areas from this plate onto fresh plates and voila – single colonies.

Hope this helps….

Philana

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tet system with rtTA

2009-08-24T11:32:19Z

I am looking for a TET SYSTEM (tetracyclin induce system) PLASMID WHICH HAS rtTA (REVERSE TRANS ACTIVATOR) for yeast expression . I would greatly appreciate any help. Thanks.

Alaattin KAYA

akaya@...

University of Nebraska-Lincoln

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mammalian cDNA inyeast expression vector

2009-08-20T15:49:42Z

I am looking for a mammalian cDNA library cloned into a yeast
expression vector. It would be from any tissue from mouse or human. I would greatly appreciate any help. Thanks.

Alaattin KAYA

akaya@...

University of Nebraska-Lincoln

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S. pombe glycosylation

2009-08-18T13:07:35Z

My name is Bret Light and I am with Bioneer. I saw on the web that you have done some work with S. Pombe. I wanted to let you know that Bioneer now has the complete haploid and diploid library sets for S. pombe now available. If you are interested in these libraries please let me know and I can help get you up an running with them.

Thanks for your time

Bret Light

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Gentamicin in YES media (Dann Huh)

2009-08-17T08:44:49Z

Dear All,

   I wonder if I can use Gentamicin in YES media
   for S. pombe (the fission yeast) to prevent bacterial
   contamination as we do for mammalian cells.

   Did anyone try before?

-regards,
     Dann

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Postdoctoral Positions

2009-07-29T08:28:58Z

Postdoctoral Positions Dear Colleagues,
I am searching for a postdoctoral fellow to work on telomere dynamics in yeast for a start date early in 2010. My major emphasis is on recombination between telomeres and the role of a regulatory region of the major repair and telomere protein, Mre11, in that process. We are also studying the mechanism of telomere rapid deletion as a model system for telomere size homeostasis. For a review of my interests, please see Lustig (2003) Nature Reviews Genetics, 4, 916.

If interested, please contact me by e-mail or at the address below.

Arthur J. Lustig PhD
Professor, Department of Biochemistry
Tulane University Health Sciences Center
1430 Tulane Avenue
New Orleans, LA 70112
ph:504-988-3688
fax:504-988-3687

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CUP1 promoter and Copper toxicity to S. cerevisae

2009-07-24T12:25:01Z

Dear Yeast Netters,
There is a question regarding CUP1 promoter:
I placed a gene of interest under the CUP1 promoter. When the cupric
sulphate is above 100 uM, I start to see the dark cell sediment on the
tube bottom. I assume this is due to the copper toxicity. When the
inducer goes up to 500 uM, I saw improved protein production, but the
cell growth was inhibited a lot, and i saw debris by Flow cytometry.
However, I checked with a paper ( Hottiger, T., Furst, P., Pohlig, G. &
Heim, J. Physiological Characterization of the Yeast Metallothionein
(Cup1) Promoter, and Consequences of Overexpressing Its Transcriptional
Activator, Ace1. Yeast 10, 283-296 (1994)), the amount of cupric
sulphate was much lower than the toxicity limit (2mM).

Would you please share with me your experience on CUP1 promoter? Is
there any substitute to replace CUP1 promoter if the toxicity is a
really problem?

Thanks a lot!

Jingjing

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yeast CGH analysis

2009-07-21T12:02:21Z

Dear colleagues,

we are interested in doing yeast CGH and I have couple of questions:

- did any of you use services of Nimblegen (Roche) for yeast CGH and how satisfied (or not) were you with the service/cost and what are your general comments?

- do you know of any lab that successfully does yeast CGH and would be interested in collaborating with us (doing the CGH for us as a part of collaboration)?

Thank you very much in advance for all your help,

Marija Vujcic

Roswell Park Cancer Institute

Buffalo, NY

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asking for information

2009-07-14T11:00:01Z

Hello,Searching the net I found a response about liquid b-gal assays, and since you have vast experience on this issue I decided to write to you. I'm using pSV-Bgal for transfection normalization, so far I didn't get good results (at least not what I expected!), and I don't know if the problem is on the way used for detection. I'm doing assays with reporter systems and I must say I don't have much experience on this. I'm just starting my work (I'm a PhD student at Buenos Aires School of Medicine in Argentina), in fact I'm doing the first activity analysis in order to find the minimal promoter sequence of a gene. I did some in silico research to learn more about the promoter, and got some "potential" promoters (using Mat Inspector). My first attempt was to take a 2000bp fragment upstream exon 1 by PCR on HeLa cells and start activity measurements. The gene of interest codes for a protein that plays a role in autophagy, and its expression is upregulated in different stress situations included starvation.
I cloned the sequence on pGL3 vector and checked activity after transfecting HeLa cells which were then treated under starvation conditions; as a control I used the pGL3 empty vector and for transfection control I used pSV40-Bgal.
So far I didn't got good activity results......I don't find consistent differences between treated and untreated cells (we suppose that in normal conditions the promoter is not going to be very active, or better said.....under starvation conditions we expect to have a great activation of the expression, and therefore have a strong luciferase signal)...so after dealing a lot trying to find the reason....I now blame it on everything!!!
I first considered that maybe 2000bp is not enough and I should go further upstream the ATG.
Then I also wonder if the detection system is ok. I'm using a Firefly luc assay kit for pGL3 activity detection, and for Bgal I do a colorimetric measurement using ONPG at 420nm OD. I rinse the cells with passive buffer and use a sample for luminiscence and other for bgal. Every time I did it, samples took a long time to turn into yellow, and some of them even didn't changed at all, so results were so variable that I'm not sure about them..Do you have a standard protocol to measure B-gal activity? I also realised you normalized with protein concentration...In this case is it enough to have the b-gal value to normalize the luciferase results? or I should also take into account proteins concentration?I know you work with yeast but...
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homologous recombination in yeast

2009-07-08T20:05:29Z

Dear Yeast Netters,
I have some questions about homologous recombination in yeast:
1. In the chromosome, I already integrated a DNA sequence (~800bp). What
if I transform a replication plasmid with the same/very similar DNA
sequence? Will the two recombine?
2. a DNA sequence (~1000bp) was integrated first into the chromosome. A
second sequence, which has more than two domains of high similarity with
the first integrated sequence. I wonder if recombination will happen. If
so, will it have more than one crossover?

Thanks,
Jingjing

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Re: yeast plates with oleic acid

2009-06-25T10:39:34Z

I have cultured yeast in wall board, and am running remediation tests.
If I need to isolate yeast species for Heat, cold envelope testing.
Or isolate for chemical testing or gel shipping purposes, do you
reccommend particular assay to separate cleanly from solid substrate
and identify? Currently we are using a modified already batched
saccharomeyes w/ecoli xylose breakdown courtesy of DoD operations here
in Santa Ana, Ca. You guys are the best! thanks Mr. D

On Tue, Jun 23, 2009 at 12:45 PM, Stephanie
Kaut<stephanie.kaut@...> wrote:

> Hello together,
>
> I want to test some yeast strains for growth on YPAD plates with oleic
> acid.
> But preparation of the plates seems to be tricky, because oleic acid and
> the agar don't mix well, and the oleic acid swims on the top of the agar.
> What can I do to have nice plates?
>
> Best regards,
> Stephanie
>
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Re: Promoters for RNA pol I and III

2009-06-24T13:11:49Z

I know the yeast three hybrid system uses the polIII promoter and they have the sequence for the plasmid posted here:

http://www.biochem.wisc.edu/faculty/wickens/lab/3h.aspx

You can also search for non-coding RNA's here

http://www.yeastgenome.org/cgi-bin/search/featureSearch

and find polIII encoded genes and look at the papers that identified them:

http://www.yeastgenome.org/cgi-bin/reference/reference.pl?dbid=S000064699

~Jp

On Jun 16, 2009, at 6:02 AM, Søren Helmark wrote:

Hi,

Do you know if promoters for RNA pol I and III are characterized and where I can find the exact sequence (databases/papers...)

Regards,

Soren
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yeast plates with oleic acid

2009-06-23T12:45:25Z

Hello together,

I want to test some yeast strains for growth on YPAD plates with oleic acid.
But preparation of the plates seems to be tricky, because oleic acid and
the agar don't mix well, and the oleic acid swims on the top of the agar.
What can I do to have nice plates?

Best regards,
Stephanie

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No background with yeast two-hybrid screen

2009-06-18T09:17:58Z

No background with yeast two-hybrid screen

Hi, All,

I was hoping that someone may have some insight into my problem as I am quite stumped. I performed a yeast two-hybrid screen using Clontech's new Matchmaker Gold system, which tests for interactions by growth on the antibiotic Aureobasidin A (AbA, 200ng/ml concentration), appearance of blue color with addition of X-alpha-Gal, growth on -HIS, and growth on -Ade. My bait was in strain Y2HGold (which apparently is very much like the strain in their Matchmaker 3 kit - AH109) and my library in Y187. I thawed an aliquot of my library in Y187 (after thawing, the titer was ~10^8 cfu/ml) and mated the library strain to the bait strain. After mating the strains, I tested the viability of the diploids (~10^5 cfu/ml), the bait (~10^8 cfu/ml), and the library (~10^6 cfu/ml) on the appropriate media and I have screened over 10^6 diploids as the company manual says to. I initially tested for interactions on the lower stringency media, which is -LEU, -TRP, X-alpha-Gal, AbA and there was absolutely no growth. I retested for interactions on lower stringency medium by reducing the amount of antibiotic to 50ng/ml, 100ng/ml, and 150ng/ml. I still have no growth even on the lowest concentration of antibiotic. I then tested for interactions on -LEU, -TRP, -HIS, X-alpha-Gal and I still get nothing! I am really surprised by this last result as everything I have read suggests that HIS expression is very leaky and needs to be suppressed by 3-AT in order to detect true interactions.

I have previously tested to make sure that my bait does not auto-activate, is not toxic, and is expressed. The positive and negative controls provided by the company behave exactly as they should so I know the media preparation is not the problem. Therefore, I am quite stumped as to why I am getting absolutely no background, even on media (-HIS) that is supposed to give high background. Any ideas on what could be happening?

Has anyone worked with the Matchmaker Gold kit and what have your experiences been?

Best,
Jennifer

Regards,
Jennifer Chang, PhD
Post Doctoral Fellow, Cell Biology
Medical Sciences Building, Room 698
New York University School of Medicine
550 First Avenue
New York, NY 10016
Tel 212–263-5316
Fax 212-263-8561

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Promoters for RNA pol I and III

2009-06-16T04:02:31Z

Hi,

Do you know if promoters for RNA pol I and III are characterized and where I can find the exact sequence (databases/papers...)

Regards,

Soren

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lysis of yeast cells

2009-06-08T09:24:38Z

Hello

I saw your previous message from 1998, an read it. I was wondering what process you use to lyse yeast cells in order to gain protein. I know that the massage below was used to gather DNA, but you stated in your reply that there is a whole different process for gaining protein and I was wondering what you do. Thanks

Kanchen

lysis of yeast cells

Rob Kirkpatrick ...
Wed Sep 30 09:03:05 EST 1998

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In article <3609213C.1101 at bio.uio.no>, Trygve Meum Eliassen

<t.m.eliasen at bio.uio.no> wrote:

> I am using glass beads (which is the method I have available) to lyse

> S.cer. cells. The method usually works eventually, but it is just so

> inefficient. After 10x1 min of vortex, only a small fraction of the

> cells are lysed. I have collected cells from 50 ml of minimal medium, OD

> 0.5, then added a small volume of phosphate buffer (or SDS-PAGE buffer),

> and glass beads (0.5 mm) to just below the meniscus.  Then 1 min.

> periods of vortex with 1 min. on ice inbetween. I have tried with

> several different tubes; eppendorf and bigger.

> So obviously I am doing something wrong. I would be grateful if someone

> would tell me what. From thomas.moen at biotek.uio.no

I can't trouble-shoot your method because it is a lot different then ours,

but I'll give you our lysis buffer recipe which we use for glass bead

preps (we call it Yeast Cracking Buffer):

2 ml TritonX-100

5ml 20%SDS

2ml 5M NaCl

2ml 1M Tris-Cl pH8

2ml 0.5M EDTA pH8

bring up volume to 100ml with H2O

We use this to obtain DNA samples for southerns and plasmid rescue in the

Two-hybrid system and it works well.   We start off with a 2ml log-phase

culture of yeast grown in rich culture (may need more volume for minimal

media).  This is pelleted and resuspended in 500microlitres of water and

pelleted again.  This is then resuspended in 200microlitres of our

Cracking Buffer and glass beads are added until a small amount of liquid

is visible.  Next we add 200microlitres of Phenol:Chloroform (1:1).  This

is then vortexed 3 times for 30 sec with at least 30 sec on ice in between

vortexes.  We spin this then remove the upper layer and precipitate it.  I

don't know the actual amount of DNA you get from this but it's plenty for

a southern.

If you're after protein, the story changes.  Let us know exactly what you

want to lyse the cells for and we can give you more specific protocals.

Rob

--

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Liquid media?

2009-05-22T15:08:33Z

Hi again everyone, and thanks for all the previous responses to my growth rate question.

We have already tried to assess yeast growth rates using OD600 in TSB (tryptic soy broth) media. We got rather spurious results, with the OD jumping all over the place between measurements. What is the appropriate liquid media to use for Pichia, Candida, and Saccharomyces?

We are worried that our previous dubious results may be due to using the wrong kind of liquid media.

Once again, any advice on the appropriate media/technique would be greatly appreciated.

Thanks again,

Seth Davis

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spores isolation

2009-05-22T07:59:58Z

Hi all,

I would like to isolate spores of S. cerevisiae from a mixed culture containing spores and vegetative (haploid and some diploid) cells. I obtain that culture by digestion with zymolyase (which, by the way, is rather inefficient at killing vegetative cells of my strain). I am currently using mineral oil and water: spores segregate in the oil and cells segregate in the water. But it is almost impossible to get the spores out of the oil. Even centrifuging at 13,000 rpm for over 20 mins won't transfer them from the oil into water. Ultimately, my goal is to transfer the spores (with almost no vegetative cells) into liquid growth medium. Any suggestions on how I can isolate the spores?

I have checked the archive from the yeast list and I couldn't find a solution to that problem.

Thank you in advance,

Jean-Nicolas

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determining growth rate

2009-05-20T18:15:40Z

Hi everyone,

I am an entomologist trying to study the microbial community associated with an herbivorous beetle species. As such, our lab is rather limited in terms of microbiological sophistication. We have tentatively isolated a wealth of yeast colonies from the glandular mycangia of these insects, and would like to conduct some performance-type experiments using our yeast isolates and chemical treatments. Our isolations are currently being done on 2% MEA, and appear to mostly be in the genera Pichia, Saccharomyces, and Candida.

My question to the group is this:

What is a reliable, quick, and easily interpreted method for assessing growth rates of yeast colonies? We are familiar with the technique of colony forming unit count via serial dilutions, however, this seems to be a laborious means of assessing growth rate over time with many strains and (potentially) multiple chemical treatments. Or is it simply best to try to obtain a single 'snapshot' of growth rate?

Any advice that could be offered would be greatly appreciated. Thank you.

Seth Davis

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query for mutant cells

2009-05-06T00:07:01Z

Does anybody have

"D18 cds1∆ ura- s.pombe cells" and

"D18 rad3∆ ura- s. pombe cells"

Kindly tell how to get

--
- Rahul Kumar Mishra
c/o Prof. D.D. Dubey
Molecular Biology Laboratory,
Dptt. of Biotechnology,
VBS Purvanchal University,
Jaunpur-222001

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query for highly transformable S.Pombe cell

2009-05-01T22:58:07Z

I am extremly sorry for my blunder in my previous mail regarding highly transformable S.Pombe vector. By mistake it was written 'Vector' instead of ' Cells '. I apologise for that.

That should be read "highly transformable S.Pombe cell" as that VL6-48N of S. cerevisiae. (ref. Natalay Kouprina, vladimir N. Noskov and Vladimir Larionov :methods in Molecular Biology, Vol 349).

--
Mukesh Pratap Yadava
C/O Prof. D.D. Dubey
Molecular Biology Laboratory
Department of Biotechnolgy
Faculty Building
VBS Purvanchal University
Jaunpur-222001 (Uttar pradesh)
Mob. No- +91-9451157085

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query for highly transformable vector

2009-04-22T00:10:44Z

My work inclueds routine transformation experiments in yeast and E.coli.
I wonder if anybody knows about a highly transformable S.Pombe vector as that VL6-48N of S. cerevisiae. (ref. Natalay Kouprina, vladimir N. Noskov and Vladimir Larionov :methods in Molecular Biology, Vol 349)
--

--
Mukesh Pratap Yadava
C/O Prof. D.D. Dubey
Molecular Biology Laboratory
Department of Biotechnolgy
Faculty Building
VBS Purvanchal University
Jaunpur-222001 (Uttar pradesh)
Mob. No- +91-9451157085

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monitor cell lysis during cell culture

2009-04-14T11:39:52Z

Hi,

I am purifying a protein that is being secreted to culture medium, however, I encounter significant amount of cytosolic proteins in the supe. I guess these are resulted from cell lysis. If it is true, I'd like to monitor how much yeast cell lysis is going on during exponential cell growth.

Please give me some advise/comments for markers, methods and references. My email is jinyohan@...

Thank you so much, in advance.

Yohan Jin

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Looking for a shuttle vector with GST tag.

2009-03-19T07:51:50Z

Dears all,

I' m looking for shuttle vector to perform a homologuos recombination in yeast S. cerevisiae genome.

The plasmid must be tagged with GST and allow the C-terminal protein tagging (Protein-GST).

Does anybody have it?

looking for having positive feedback,

a nice day
Cinzia

Pagliuca Cinzia, PhD.
c/o Campus IFOM-IEO
via Adamello, 16
20139 Milano, Italy.

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Lab tech position available

2009-03-16T12:07:12Z

A Research Support Specialist position is available in the laboratory of Dr. Joshua Rest in the Department of Ecology and Evolution at Stony Brook University.

Brief Description of Duties: Perform large-scale serial growth competitions in yeast.

Perform large scale serial growth competitions, including regular serial dilutions, relative fluorescence measurements, and associated data collection.
Construct and validate yeast strains; design primers; transform strains.
Perform ancillary competition validation: flow cytometry; large-scale RNA and DNA isolation; pyrosequencing.
Data analysis and integration.
General lab maintenance.

Salary: $34,975 - $45,200
Required Qualifications: Bachelor's degree. Ability to demonstrate professional competence in research activities. Course work and laboratory experience with molecular biology protocols and equipment.
Preferred Qualifications: Two years of laboratory experience either in yeast genetics or in high throughput biological analysis, such as cell culture or expression microarrays.
For additional information on the Department or its activities, please visit http://life.bio.sunysb.edu/ee/restlab/index.htm.

For more information about this position, or to apply online, please go to:
http://tinyurl.com/labtech
or
http://naples.cc.sunysb.edu/Admin/CampusJob.nsf/ebb5a78b9e8848868525659c0072eafe/1b090b4ec1c469f1852575710062a1f4?OpenDocument

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slower growth rate of transformants

2009-03-08T01:07:39Z

Hi, there,

Does anybody know the casue of a much more slower growth rate of transformans? we introduced a 5kb heterologous ABC transporter gene in to Saccharomyces cerevisiae single mutant for a complementation experiment( integration vector, URA3 selection), the transformants did show up on selective medium but stay as a very little tiny colonies even after incubation for up to one week. any trouble shooting tips on this issue?

Thanks in advance.

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Looking for an explanation

2009-02-25T11:13:49Z

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

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