Science topic

Yeasts - Science topic

Yeasts are eukaryotic micro-organisms which belong to Ascomycota and the Basidiomycota phylogenic groups. 1,500 species have been currently described and are estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding.
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Hi, I need to check microbial growth on wastes rich in proteins that tends to ferment pretty quickly. To get an idea of what and how much it's growing, I thought about using Plate Count agar, YS or YM for screening for yeasts, maybe Violet Red Bile for gram negative. What can I use to check protease activity? Since it's a mixed colture, I cannot use super selective media.
Could this course of action work for a totally uknown mixed colture? The goal is to identify the microrganisms responsible of spoilage and fermentation.
Thanks in advance for the help.
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Bacteria frequently are identified by sequencing ribosomal RNA. Thus, NGS could be a viable approach.
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I am looking for a methodology for lab scale extraction of wax esters from bacterial or yeast cells
Any input would be apprciated
Thanks in advance
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Jakub Grzesiak Can you cite any reference for the method you mentioned? thanks
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Dear yeast biologist,
I performed 4-5 time yeast transformation in S. cerevisiae SK1 MATalpha strain but I am unable to get transformants.
I performed and checked all the necessary steps as per my knowledge.
If possible please suggest me, how to troubleshoot the problem?
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It would be easier to understand your trouble if you mention which type of protocol you are following and which type of DNA (plasmid or DNA cassette, its size and amount), selection marker (antibiotic or auxotrophic) etc. with your question.
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I will transform the plasmid that contains the gene of interest. The plasmid will be transformed into yeast cells by electrophoration. The yeast used comes from isolated baker's yeast to obtain a single colony. is it possible for the plasmid to enter into yeast cells? or do I need a competent yeast with a specific strain specifically used for transformation? Please help me.
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The problem is whether you have a selection for the plasmid. The normal lab strains of S. cerevisae are often auxotrophs that are used for plasmid selection but a wild type strain would not have these markers to use. Note that bakers yeast is also still S. cerevisiae but just a notably different strain than the domesticated lab versions.
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I am examining the presence of lactobacillus in a commercially available kombucha sample. Following serial dilution upto 10^3 because above that dilution no growth was observed. I had tried the experiment multiple times followed by gram staining I get red rod shaped bacteria and dark purple yeast. Can any one give the reason why?.
Kombucha pH-2.7
Incubation period - 48-72 hrs at 30•c in a candle jar.
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Have you run Gram stain controls for your procedure? Kombucha includes yeast.
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I have been trying to stain my yeast cells with DAPI after fixing them with 4% PFA and I am not getting a good result. Maybe someone has a tip they can share on how to do it to get nice pictures!
Also, is there a way to use DAPI staining in cells in stationary phase without having to add additional glucose to the media??
Thanks a lot for your help!
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During DAPI staining of yeast cells, the cell wall of yeast cells also get stained. I have also used 0.1% Triton X. But still getting the same problem.I am using 1μg/ml DAPI. I also have increased the washing. Please suggest any solution for this.
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Hello,
I recently found out a contamination in my cell culture.
However, I would like to ask, what could it be, exactly???
Contamination initially does not change the color and pH of the media, this happens after about 5 days.
According to the microbiological agar smear, it does not seam to be mold.
Could it be yeast contamination?
Thank you for the advice.
Have a nice day.
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Hello,
The contamination of your culture may be type of fungus.
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Hi all,
I am looking for yeast selections that result in adaptive mutations appearing only in a single gene. That is, if I pick single colonies that survived this specific selection, all or nearly all of them will have the adaptive mutation/s in the same gene. 5-FOA selection, for example, results in adaptive mutations mostly in the URA3 gene. The same for canavanine and CAN1. I am looking for more such examples. I am not looking for solutions that involve genetic engineering (i.e., no selection for revertants of introduced conditional deleterious mutations etc.).
Thank you
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Thank you Hanna,
This is a great example. I wasn't aware of it.
Daniel
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Because some researches do autolysis of cells first, then treatment with the base, and others do the treatment with the base directly, which is better and why?
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think you'll get a lot of cellular protein with whole cell extraction.
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I would like to present isolated yeast genome comparative with S. cerevisae S288c and present on circular visulization. So what's program and how did it. ?
Ps. I'm biologist and haven't basic about phyton or R program.
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Looks like something from circlize package in R. I do not work with yeast. While such a chart might be highly meaningful to another yeast genomics expert, it really looks like abstract art to me. Too much information condensed too compactly to be able to extract even the simplest information. In an oral presentation it might take me a couple of hours to walk the audience through such a graphic to actionable results.
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Hi everyone,
I've been recently culturing 4T1 cells. The cells looked happy and healthy after thawing for a few days (i changed the medium every other day); however, after trypsinization, they had abnormal morphology and all died after a day. I've attached two images of the culture just before throwing them away. The culture was also a bit bad-smelling and somehow turbid.
I had the same problem with my other cell line NIH3T3. The cells are in good health initially but develop small particles and brownish clusters after trypsinization, and die after a day or two.
I assume this might be a contamination problem, probably with yeast.
Can anybody tell me what type of contamination they think this is, and how we can get rid of it for our future cultures?
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In my opinion the photographs give an impression of yeast. In order to confirm whether this is yeast, you may please inoculate the suspected culture on Sabouraud dextrose agar or Pal sunflower seed medium ,and study the macroscopic as well as microscopic morphology of the suspected yeast colony. Pal sunflower seed medium was developed by us in 1975 as a selective medium for the rapid isolation and presumptive identification of Cryptococcus neoformans from clinical and environmental materials.Now this medium is widely used for the isolation of yeasts and also non-dermatophytic filamentous fungi, such as Aspergillus, Fusarium, Alternaria, Curvularia, Mucor, Rhizopus etc.
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For my Y2H experiment, my yeast gold strain transformed with an empty AD vector (PGADT7) is growing on DDO medium (not growing with empty BD). What could be the possible reasons and how to figure them out?
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if you wanna help you got to be more descriptive...
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I have a collection of bacteria + yeasts from plant/soil origin. I need to eliminate yeasts while retaining bacteria. What would be the most accurate and economical method to use?
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Hi there,
Centrifugation is very effective as yeast is very dense compared to bacteria. For instance you can pellet S. cerevisiae just by spinning your sample for 1 minute at 1000g while you need at least 10 minutes at 6000g to pellet E. coli...
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I used two expression plasmids, pESC-His and pESC-Trp, to express 4 genes in Saccharomyces cerevisiae. Initially when I expressed these genes, I found that the strain was producing the compound of interest, a sesquiterpene-alcohol. About one year later I had to repeat this experiment, so I revived the strain from cryo stock and repeated the fermentation as before. Surprisingly no compound was produced. I checked all the chemicals and media components used and didn't find a problem. Finally, I retransformed the yeast with the expression vectors to obtain fresh strains. More surprisingly, the compound is still not formed in these fresh strains. I would really appreciate anyone who can help me with this problem. Thank you
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I find hard to believe that all the cells in the stock would stop working. It is easier to explain it with a mistake in labeling the stocks.
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SInce Notch is a cell signalling pathway, and yeast is unicellular, does notch exist in yeast?
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Hello Umang,
Notch signalling is an evolutionarily conserved pathway present in some organisms, such as drosophilas and humans, however some studies indicate that this signalling pathway may be involved with the infectious process of some pathogenic fungi, as well as modulation of the immune response against these fungi.
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Hello! I am wondering if I can use saturated yeast cultures for a spot plate assay a week after storing them in the fridge. I am also wondering if I can use yeast cultures in log phase (~1x10^7 cells/ml) for an experiment a week or a few days after storing. Will they maintain log phase in the fridge? I am working with Saccharomyces cerevisiae.
Thank you!
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I am wondering if I can use saturated yeast cultures for a spot plate assay a week after storing them in the fridge.
Yes, you can. You may store your liquid culture for a week in the fridge (4ºC). But for a spot plate assay, you will have to make a new starter, and wake them up again.
I am also wondering if I can use yeast cultures in log phase (~1x10^7 cells/ml) for an experiment a week or a few days after storing. Will they maintain log phase in the fridge?
No, they won’t.
Yeast is fussy about temperature. The growth rate of yeast varies with temperature. Yeast grows well at room temperature, but they grow more rapidly at 30-35˚C. Well-aerated cultures grow more quickly than those that are not, so liquid cultures are usually grown on a rotary shaker. Therefore, they will not maintain log phase in the refrigerator.
Best.
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Hello everyone. I'm having a huge problem with glucose measurements. I want to do a glucose consumption profile along a yeast full fermentation, from exponential to stationary phase. Right now, I tried both HPLC and a glucose assay kit (glucose oxidase based). I did the controls (different glucose concentrations in my media without yeast) which are ok - there is linear correlation concentration/OD for the assay kit, and the HPLC measures accuratly. However, on my fermentation samples, the concentration never goes down than 20 g.L, even at the end of the fermentation, and the values along fermentation are very erratic (zig zag, 30 to 40 to 20 to 30 g.L), with both methods. I tried different sampling procedures (either sample filtration or centrifugation or a combination of both, with HPLC certified filters) and nothing works. Im sure that glucose is my limiting reagent, because I did some growth tests with different glucose concentrations, where cells cease growth proportionally to glucose concentration. Can't think about any other reason for this to happen. I hope someone can help. Thank you all 🙂.
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Hello Paul, thanks for your advice, I'll see what I can do.
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Hi there,
After a good previous experience using the O-GlcNAc-Specific Antibody CTD110.6, WBs do not work anymore. In particular a negative control (previous used) and a new negative control (recently added to our experiments) are both recognized by this antibody.
Here goes my conditions and a WB picture of the problem, in case someone can help.
Thanks in advance ,-)
- Nitrocellulose membrane (Pall) 0.2 µm.
- Blocking 1h RT in TBS 1X + Tween 20 (0.1 %) + BSA (Bovine Serum Albumin Fraction V, Merck) 10 %.
- Primary Ab --> Anti-O-GlcNAc CTD110.6 (Sigma) (1/2000) in TBS 1X + Tween 20 (0.1 %) + BSA 10 %. Incubated ON (4 ºC).
- Wash 3 times with TBS 1X + Tween 20 (0.1%).
- Secondary Ab --> HPR conjugate IgG Sheep anti-Mouse (Amersham), diluted 1:10 000 in TBS 1X + Tween 20 (0.1 %) + BSA 10 %. Incubate 2h (R.T.) Same, or even worst, results are obtained using a more specific secondary antibody (m-IgGκ BP-HRP (sc-516102) diluted 1/2500, incubated 1h at RT).
- Wash 3 times with TBS 1X + Tween 20 (0.1%).
- Chemiluminescent development by Clarity ECL Western Blotting Substrate.
Two types of negative controls have been included.
(1) Plant virions unable to O-GlcNAcylation
(2) Total protein from yeast (S. cereviceae), OGT lacking organisms. Specially for yeast a null O-GlcNAcylation signal has been reported in several publications.
In both cases, no signal is expected. However, WB shows an CTD110.6 over-recognition and negative control failures.
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Thank you very much Chandru, I noted your recommendation. I'm afraid the background trouble is a general problem. Thanks again.
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Right now, I am doing a project to characterize yeasts that I isolated from honey.
I found that the yeasts can grow even at 40% sucrose medium. is this yeast can be considered osmotolerant yeast, if yes I needed some reference to prove that this yeast are may or in fact osmotolerant.
Thanks.
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Try to find earlier reports on similar line, you should fine information regarding their standard classification based on concentration of sugar uptake
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In bio-catalysis reaction is going on with yeast and Chloro-Aldehyde in Lb media ,which solvent might use as mobile phase?
My mobile phase is methanol:water , can i put directly inject sample to HPLC or need any treatment.
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Maybe hexane and iso-propanol would be good flowing phase.Of course,they should be mixed in a certain proportion.Acid condition would help keep something stable.
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I'm doing yeast two hybrid screening. When I found potential interactions, I did plasmid rescue and cotransformed with my bait plasmid empty vector as a control (rescued prey+bait / rescued prey+emptry). After transformation I spread the transformants on SD/-Leu/-Trp and then streaked the yeast colonies on SD/-Leu/-Trp/-Ade/-His to verify interaction.
My problem is that when I streaked 10 yeast colonies of each rescued prey+bait and rescued prey+empty vector on SD/-Leu/-Trp/-Ade/-His, some of them (rescued prey+bait) which were expected to grow on SD/-Leu/-Trp/-Ade/-His were not grown and also some of them (rescued prey+empty) which were not expected to grow on SD/-Leu/-Trp/-Ade/-His were grown. Can anyone explain about this problem? Thank you any help.
(I also checked my bait and empty vector were transformed correctly using yeast colony pcr)
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I understood your question.
First, test your regents using some positive controls (well-characterized bait and prey interaction in your lab).
Second, in addition to rescued prey+empty, use bait+empty transformation to rule out self-activation transcription factors by bait proteins.
Third, instead of streaking colonies only on SD/-Leu/-Trp/-Ade/-His, you can also streak on SD/-Leu/-Trp/-Ade and SD/-Leu/-Trp/-His, SD/-Leu/-Trp/-Ade/-His+5mM 3AT, SD/-Leu/-Trp/-Ade/-His+10mM 3AT & SD/-Leu/-Trp/-Ade/-His+20mM 3AT. In this way, you can have many controls, and it would be easy to troubleshoot and interpret the results as well.
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My compounds is choloro benzaldehyde and it is insoluble in water , i am willing to do biotransformation in LB (Lysogeny Broth )media , what technique might be useful which doesn't affect yeast cells also?
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I suggest ethanol or DMSO.
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I'm inserting a gene piece by piece into a plasmid because of certain changes I need in the sequence. Once the gene sequence is complete in the plasmid, the yeast appears to recognize the gene, and "repair" it to the original sequence.
I have verified this using sequencing and PCR (the constructed gene has restriction sites at the seams, the sequences of which I use as promoters for the verification PCR).
Is there a way to stop this process, or to slow it down, so the modifications I have in the gene will remain there?
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Sadly we don't have any such strains in our lab.
Can you name any such mutant strains so I could ask around in other labs that work with yeast?
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Zobell marine broth or simple Glucose yeast extract with peptone
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sea water medium
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Hi all,
I'm setting up a yeast display for measuring Kd of a small molecule tagged with FITC binding to our protein of interest expressed using a standard yeast display protocol. I wonder what is the Kd detection limit for yeast display. Our protein is expected to bind the ligand with 30uM Kd. Given there are multiple wash steps, would those with higher k_off binding small molecules fall off the protein? Thanks!
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I've never tried to measure a Kd this way, but from other experience I've had, my expectation is that a substance with a 30 µM Kd would be lost during washes.
Another point is that a Kd measurement is an equilibrium binding measurement. If there are washes involved, it cannot be an equilibrium binding measurement, and therefore whatever is measured will not be a real Kd.
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Hello everyone,
I am trying to optimize fermentation conditions in my enzymatic hydrolysate obtained after cellulase treatment. I am using S. cerevisiae MTCC 36 to carry out the experiment. Even though this strain has previously reported a high ethanol conversion rate, I am only getting 0.7% ethanol yield in a solution containing 13.4g/L of glucose. I am carrying out the experiment by supplementing the enzymatic hydrolysate (1L) with yeast extract (10 g) and peptone (20 g) adjusted to pH 5. Incubation is carried out at 30 degrees with 150 rpm in a baby modular fermenter. My culture volume was 300 ml and the fermentation rate started decreasing after 6 h.
I hope this is enough information. Thanks in advance.
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8/8/22
Dear Najya,
Consider:
1 mole Glucose -> 2 moles EtOH + 2 moles CO2
Your sugar concentration is 13.7 g/L, or 74.4 mMoles glucose. Given the above stoichiometry, your maximum theoretical EtOH yield is 148.9 mMoles.
149 mMoles EtOH x 46 mG/mMole = 6854 mG EtOH, or 6.854 g/L. This is 0.6854 g/100 mL, or 0.685%, which is just about what you are getting. If you want a higher yield of EtOH, you need to add more sugar to the fermentation.
I hope this information helps you.
Bill Colonna, Iowa State University, Ames, Iowa, USA wcolonna@iastate.edu
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Hello, researchers
I used sodium benzoate as a preservative while working on improving the shelf life of beverages. It inhibited yeast from growing while promoting the growth of lactic acid bacteria. What could be the reasons?
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Actually, as the salt of any other acids, sodium benzoate may inhibit the growth of lactic acid bacteria depending on its concentration. Here, it just eliminates yeasts regarded as their competitors, it can achieve more nutrients to be grown.
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This is a primary glial culture (taken from pups @PD2). Picture taken at 10DIV. Astrocyte layer is completely confluent. We are actually after the microglial layer underneath the astrocytes (Will use Saura et al. mild trypsinization procedure at DIV15). I'm thinking that the smaller cells might be yeast, except some of them have small projections. Thoughts? We are very strict about our cell culture protocols - all culture work is done in a BSC that is rigourously sanitized with 70%ETOH. Anything brought into the hood is sprayed/wiped with 70% ETOH, so not sure how yeast would get into this culture? The only thing I can think of is that during initial harvest, we don't have a downdraft table - it is done in the cell culture room on a counter that is wiped down with 70% ETOH and all instruments are soaked/dried prior to use. Could it be coming in via initial harvest?
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Hi,
The things on red arrows do not look like yeast. Yeast should be smaller than these. It is possible that the culture is mixed with other cells.
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Hello, sorry if this is a naive question as I do not normally work with yeast. I am looking for a protocol that outlines how to extract/collect yeast cell walls.
Thank you,
Karen
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If you're seeking to obtain entire cell walls, you might look at physical means - bead beater, etc. with the intent that subsequent washes would remove other cellular constituents. Application has included validation to argue absence of these other cellular consituents. Think one was lipase activity.
I'm not aware of a solvent that would selectively "extract" only cell walls.
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I am having difficulty in obtaining transformants, the positive control plasmid pKLAC2(malE) was prepared for transformation and only small colonies were observed. These did not secrete malE protein. I am worried that my competent cells are bad and I am reaching out for any tips, tricks and advice that you might have for me to determine if they are good or not. I will try to summarize my concerns, observations and questions. Q1: Are transformants on YCB (yeast carbon base) + acetamide (sole nitrogen source) expected to grow similarly to K. lactis on rich media? Even when I plate a very small volume of the transformation reaction, I get >300 small (0.1 mm) colonies after 24 hours. They proceed to grow slowly and I am aware that untransformed cells have weak acetamide degrading ability. I have subcultured a few select colonies and they grow fine on YCB + acetamide, albeit half as well as colonies growing on rich PDA media. They must not be transformants? Q2: Untransformed cells appear to be growing better than they should be....could it be possible that impurities in agar could be supplying nitrogen for growth? Small colonies after 24 hours can't be normal but I can't determine what is weakening the selection method. Or selection by acetamide is not as selective as I thought. Q3: After adding the transformation reagent to competent K. lactis cells, they remain in small (0.1 - 0.4 mm) visible clumps and aggregate. Is this normal? The solution never quite reaches a uniform turbidity, even after the incubation, heat shock and recovery steps. Q4: Unrelated question, galactose wouldn't go bad would it? I have a bottle from at least 30 years ago. I know that glucose has an indefinite shelf life (pretty much) but I'm not certain if "bad" galactose would explain the absence of protein expression.
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You should compare colony numbers between your selection plate and a rich media plate inoculated with the same volume from the transfection. If numbers are similar your transfection has failed.
Depending on your rich media you should find that there is always a slight reduction in growth on poorer media.
Any of your reagents could be contaminated. Could even be nitrogen fixing bacteria if any of your powders got damp.
If kept dry the galactose should be fine if not exposed to sun light.
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Hi, Im searching for a yeast mannan resin. Does anyone know where I can order from?
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Familiar with yeast mannan but not yeast mannan resin. What is that?
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Why does MAT A type can only be crossed with MAT alpha type in yeast?
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Because MATa cell is specifically expressing a receptor specific to alpha factor. Interaction between the receptor and the pheromone is the initial step of a signalization cascade leading to the "shmoo" phenotype (cell stopped in late G1 physically waiting for its complementary mating partner (ie. MATalpha cell conditioned with the a-factor) for cell fusion.
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I'm currently confused on the centrifuge speed needed for separate yeast cells (Saccharomyces cerevisiae) from its aqueous medium
I need the yeast cells to still alive to ferment herbal extracts
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Thank you for your answer sir
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Dear all,
I inoculated a 200 ml minimal medium with 50g/L glucose with an unknown yeast (shown in figures).
After an incubation of 72 hours, at 180 RPM and 28 C, the broth containing the yeast turned very viscous, slippery liquid. In HPLC, using H column with 5mM Sulphuric Acid as mobile phase and RID as well as UV detector, nothing was detected.
The colour of the yeast is not cream or white, but its like cantaloupe colour.
Any type of help in identifying this yeast or the viscous product shall be highly appreciated.
Best Regards
Arush
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@Arushdeep, I also recommend molecular identification.
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After treating the cells, I'll be sending my samples to another organization for performing RNA-seq. RNA extraction will be done by them. I have a large number of samples and to prepare each sample, it will take 1 week time. I do not need live cells at the end of this exercise. I want to know for how long I can store them, so that I can send multiple samples together, saving time and other logistical hassles.
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You may store your cells in RNAlater which is a RNA stabilization solution that helps to stabilize and protect RNA in samples. It eliminates the need to immediately process samples. The sample can simply be submerged in RNAlater solution and stored at -80 degree C for analysis at a later date.
Wash the cell pellet in PBS and then add 5–10 volumes RNAlater solution and store at -80 degree C.
For more information you may refer to the link below.
Best.
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I am processing some organic wastes with bacteria and yeasts I want to stop the fermentation after I get the end product, can I depend only on PH, or boiling or add some chemical to stop fermenation to preserve the final product for months.
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Dear Kasem,
It all depends on the physico-chemical characteristics of your molecules of interest.
If I understand you carry out solid fermentation, therefore, you can "stop" the fermentation by modifying the pH (pay attention also to the impact of the pH on your molecule of interest but also you risk weakening the microbial walls thus releasing other molecules that you will have to eliminate later). The temperature is another factor also with the same remarks as for the pH.
But if you want my own opinion, I will focus on the first stage of purification in order to recover, in part, your molecule of interest if it is extracellular via solvents for example or salting out. If your molecule is intracellular you will have to consider weakening the membranes of your micro-organisms via physical, mechanical or chemical processes such as solvent, pH, or detergents.
Best regards
benoît.
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Hello!
I am curious whether I can culture mammalian cells and yeast cells in the same culture room. Our lab has one cell culture room (walls are made of something similar to glass, not actual walls) that is the shape of a rectangle. Inside this room, there is another small room with its own door.
We would like to culture yeast and mammalian cells, but we are scared of contamination of the yeast onto the mammalian cells. For the yeast culture, I wouldn't be using a biosafety cabinet, and they would be cultured in a separate incubator.
I was planning to use the small room or yeast cell culture, and the bigger one for mammalian cell culture. The culture tools wouldn't be shared in any case.
We are the most worried about airborne contamination. For example, we are scared that if I handle yeasts first in the small room and then change my gloves to handle mammalian cells, some yeast cells on my lab coat or my skin would contaminate the mammalian cells. Or also when the door between the two rooms is open when workers come in or out.
I would like to know if this kind of contamination is possible, and if, with aseptic practice and maintaining tools isolated we could still culture them in the same "room". Also, may an HEPA filter/air purifier solve the problem? Any suggestions and solutions are more than welcome!
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Saccharomyces cerevisiae is not like the molds, it doesn't produce spores that would travel through the air. I worked years ago in the same biosafety cabinet with yeast cells (of Candida albicans, which is pretty similar to S. cerevisiae) and never had contamination problems. Plus, you can clean the biosafety cabinet and use the UV light in between experiments. So, I would not worry about that.
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Hello,
Lately I've been finding it increasingly difficult to find antibodies that are specific to yeast proteins (S. cerevisiae) and the usual antibody vendors (Abcam) have a very sparse yeast-specific catalog, are there any vendors that specialize in yeast antibodies or have a more extensive catalog?
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It depends on your target protein, of course, but LifeSpan Biosciences (http://www.LSBio.com) and Santa Cruz Biotech (https://www.scbt.com/) offer a fair number of yeast-specific antibodies.
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Hi guys, I hope you all are doing well
I just wanted to know wheter i can publish an experimental protocol design based only in previous literature (I have not performed it experimentally). If so, could you recommend some journals which I could submit my paper? It is a protocol for studing the intracellular and extracellular responce of human neutrophils against a yeast.
Thank you in advance
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It is difficult to publish a prposed protocol, framework or even a model in any journal without at least a pilot experiments. You should proof the applicability and efficiency of your concept with some results.
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hello dear research fellows im in a dire need of optimized protocol for human pathogenic yeast DNA extraction protocol
i have used total 700 CTAB/10% SDS as cell lysis solution along with glass powder for crushing and incubating at 60c for 15 min then adding 20ul Protinase K solution and incubating at 60c for 45 minutes then wasing the cell suspension with cholorform-isoamyl Alcohol (24:1) 2 times after this adding RNAse A solution upto 5ul for 3 hours at 37c and after it adding chilled isopropanol in equal amount and leaving at 4c for overnight next day washing with ethanol n storing with TE but still getting RNA sedimentation at the end please help my PHD work is getting affected by it. thanks in advance
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Hello Hira Ejaz
A gDNA extraction method, the glass bead Chelex 100 preparation (GC prep) method has been developed by the investigators in the attached article as an alternative to colony boil and phenol- chloroform-isoamyl alcohol (PCI) DNA extraction methods.
Yeast cells from colonies or liquid cultures are lysed by vortex mixing with glass beads and then boiled in the presence of a metal chelating resin like Chelex 100 resin. By boiling the lysate with Chelex 100 resin, cellular components are denatured and the Chelex 100 collates polyvalent metal ions, thereby reducing the activity of any nucleases which require them as co-factors.
You could use the GC prep method which has been developed to be a fast and easy way of extracting gDNA from yeast. If you are interested in this method, you could refer to the article attached below for a detailed protocol.
Best.
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Hello All,
I have been noticing that my cells haven't had the greatest viability. Typically when I passage my cells I see upper 90% in terms of viability and only see a drop in viability 3 days after I have reached terminal volume. However, recently I have seen lower viability when I passage around upper/mid 80% and then seemingly at random my cells viability drops into low 60% with very little cell density ~1e6. My first thought is contamination so I do the smell test and don't smell any sense of yeast. I check under the microscope at 4X, 10X, 20X and I don't see anything out of ordinary also checking with GFP lens and see that the cells that are clumped together are still showing green color. I check the images that are taken by the vi-cell and don't see anything out of ordinary. I have also been checking the culture visually and don't see anything that catches my eye. What else can I do to figure out what is happening with my cells?
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Hi Bianaka
So
Viability is dependent on several factors
What is the cell under culture
- recommend not performing the smell test , that may cause contamination
- Do you see contamination in culture?
- Are you providing the essential requirements, in terms of medium with its required additives and change once in 2-3 days with appropriate volume .. 5 ml minimum for T25, 15 ml for T75 ?
- how do you estimate viability?
With countess?... Cross check that by manual method
- what is the passage number?
- using non expired reagents?
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I have read some articles about oleaginous yeasts isolation but I haven't found a research which explains that these yeasts are isolated from fermented cacao beans. Is that possible to oleaginous yeast to be found in fermented cacao beans? What are the factors?
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You need to try to isolate, for a better solution.
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I am trying to perform a biofilm assay using the yeast Cryptococcus neoformans but in the protocol I found they use Dulbecco’s modified Eagle media (DMEM) which, I do not have. I have been trying to find the composition but has been impossible so far. Is this broth rich or minimal media? Which kind of media can I use instead? Thanks in advance!
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You can use minimal media, as in this article about biofilm formation by Candida albicans:
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Has anyone faced contamination from media containing Yeast Extract and Tryptone while working with SDS-Gel of proteins from a culture supernatant? If yes, what were the molecular weight of the bands and any idea about the nature of the protein showing on gels?
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Jorgaq Pata Thank you for your suggestion.
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I would like to perform some microscopy experiments on yeast zygots.
Unfortunetly zygots are clumping together which is not ideal condition for my experiment.
I tried to add 0.1% of Tween80 with vortexing and I also tried in a sonication bath.
Is there any way to avoid yeast zygots clumping?
Thank you
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Hi Hamid.
It's Saccharomyces cerevisiae. They are clumping because it's a mix of Mata and Mat alpha.
I have the same issue from both single colony and liquid medium.
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Hello! I am looking for a yeast-2-hybrid kit to buy for testing protein-protein interaction. By kit I mean something that comes with the vectors, the controls as well as the yeast strains. I have been using the LexA and ACT2.2 vectors and L40 yeast strain but since I'm facing some issues with the system, I was planning to buy a similar kit from a trusted source/company. I want to mention that I'm not doing library screens but just want to test protein-protein interactions between different proteins.
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takara or clontech is good (the matchmaker system)
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This is our first time handling this Caco-2 cell line. We thawed the vial that we had, and the cells had been growing slowly. There was a week when there was confusion with the medium used, and they were around 5 days without any supplements at all and no antibiotics. After those days, when we analyzed the cells there were these small fragments around in the culture, could this be yeast contamination or could it be cell debris? And is there a solution to this problem?
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These are not happy CaCO-2 cells.
As such they cannot provide you with what you seek. It is a false economy to save them; they are no longer related to your master stock sample. Discard and recover a fresh vial.
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Can anyone tell me is this a contamination or just a cell changing its morphology?????
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What is the cell being cultured? It looks more like a dividing cell/adherent cell changing its morphology: fibroblastic like.
The seeding density seems less
what is the displayed magnification?
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I am working with yeast lipids. After LCMS-IT-TOF and MS-dial analysis I got the list of lipids. i need to know which of these lipids belong to yeast. I was looking for a database that can help determine list of yeast lipids. I found yeast metabolome database , I guess that might not be the appropriate one.
Thank you.
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Thank you. It was helpful
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Hello! I am currently working on analyzing the genome of a yeast, Blastobotrys adeninivorans, for metabolic pathway work. I have found a complete genome sequence of the yeast here:
BUT, all the methods for auto-annotation, such as KEGG and TIGRfams, need an amino acid sequence rather than a genome. So, does anyone know of a software or method for translating eukaryotic genomes into their amino acid sequence? or for annotating eukaryotic genomes from their base pair sequences? thank you so much!!
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Hi Kathryn,
The predicted proteome of this yeast seems to be already available via JGI Mycocosm:
Maybe you can try downloading the protein sequences from there.
If you still prefer to annotate the genome from scratch, I recommend the programs BRAKER2 + AUGUSTUS for gene annotation (which can give you also the predicted proteins).
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For bioethanol production brewers yeast is used. But which commercial yeast is preferred for higher productivity?
Thank you
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Saccharomyces cerevisiae
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Zymo sell kits for yeast plasmid isolation, however I have read that you can basically just digest the yeast cell wall separately (with zymolyase treatment), then just use a Qiagen miniprep (for E. Coli) for the next steps. Was wondering if anyone had any experience using either, and if there is much difference between both approaches as they both seem to use zymolyase + alkaline lysis.
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Both works best but I would prefer zymo.
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Hello All,
I am writing this post as I don't have any prior knowledge in Protein Biochemistry and Structural Biology. I am trying to express a sub-cellular membrane protein in Pichia pastoris (strain SMD1163 his4). I have two constructs which have N-terminal and C-terminal EGFP tagged along with the protein. I have transformed the Pichia with the two constructs (linearized) and after induction with methanol, I have failed to express the protein in small volume/large volume cultures. I have following queries and any leads would be appreciated:
1. What might have gone wrong that the protein is not getting expressed?
2. Would it help to try a different strain of Pichia Pastoris (it seems Pichia is a very robust yeast which produces huge quantities of recombinant protein)
3. Can anyone have a look at the plasmid map of the constructs and suggest if I am missing something in it which is preventing its expression?
Any other ideas or ways that one can pursue to get the protein expressed would be highly appreciated.
Thank you
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Hai Li Thanks for your suggestion. I have tried to express the construct with EGFP in the C-terminus as well as N-terminus but failed.
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Hello, I made deletion of a gene in the yeast Pichia Pastoris, by the assembly method, which includes a recombinant plasmid with resistance to kanamycin and G418, for the selection of mutants. Now I have the resistant mutated strains but I would like to remove the resistance genes from them. Does anyone know of a method?
Thanks ; )
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Hi there,
Instead of antibiotics resistance cassette, you should use auxotrophic selection (URA3, TRP1, LYS2, MET15) for which counterselection exists.
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Hello, I am trying to replicate the knockout of the cytoplasmic catalase production gene CTT1 as done in this paper: .
I use the same primers for homology to the genome, but instead of using the NEO gene I amplify the KanR selection gene from the yeast toolkit (pYTK077):
I have tried selection of both 200mg/L and 500mg/L G418 YPD agar plates, in both cases a lawn grows, and colony PCR shows that the CTT1 gene remains perfectly intact with no G418.
If you have any experience with this sort of problem I would be grateful for any help/solutions/advice.
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Use less cell for transformation. Spreading a few dilution series onto Plates with G418(300mg/l will work). Incubate more than 4 days. The true KO colonies will stand out by far larger than lawn growth.
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Hi,
I have been looking into best Reference gene in Saccahromyces cerevisae (yeast) for Gene expression study RT-PCR especially to study along side URA3 gene as my gene of interest however literature have several. please input your thoughts on it?
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Hi Fg!
See this paper.
I would say ALG1 is the most reliable.
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I'm doing a yeast display experiment and have been sorting yeast cells (S. cerevisiae) with a BioRad cell sorter.
In the later rounds I've started getting contaminants on the plates. Some plates do not have any contaminants at all. In other instances it's quite possible that the contaminant is being passaged along with the sorted cells (although I'm gating a double positive population...)
The colonies have a raised margin and in some instances a "button" in the middle. The attached pics have a 200 ul pipette tip for scale; there are also some typical S. cerevisiae colonies visible in the images.
Details:
Plates: synthetic complete - trp, dextrose, pen-strep. ~2 weeks old and stored at 4C.
Growth: 2 days at 30C. Incubator is also used for E. coli plates.
Yeast strain: S. cerevisiae BJ5465
Plasmid: gal promoter so there should be no cell surface display of the DARPin library.
Cell sorter: BioRad S3. The sorting chamber is not sterile. Only yeast cells have been sorted recently although some Corynebacterium cells were analyzed in the same time frame (different days though) that yeast were sorted.
Thanks for any ideas!
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Cual es el proposito de su investigacion?
Si es su trabajo con la cepa, deberia repetir el experimento evitando la contaminacion de muestras
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I have performed several assays with the takara two-hybrid system for yeast. I cloned a small 14KDa protein as bait into pGBKT7 and I used a commercial library from takara as prey. In these I find preys/interactors that have the capacity to activate the Gal4 system in the presence of any pGBKT7 (without bait, with negative control lamine and with bait) but not with other constructions.
Bait (17KDa) + Prey : Blue
Empty + Prey: Blue
Lam + Prey: Blue
Another Bait (3 with different KDa): this one does not grow
I found that many sticky, highly charged or stringy proteins (excluding degradation pathways) can appear. But none of these proteins appear to me in other tests with different baits. Also check if it was due to protein size: no.
Does anyone have any ideas?
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Sorry, I'm talking about reporter plates. Cells in rich media can grow as usual. All the prays that I assay for my protein are active with empty plasmids o negative control plasmids with binding domine. But if I try to use another different bait preys don't work. We think that is a false positive but we don't understand why using other bait it doesn't work.
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Hi,
I want to check interaction strength between my protein of interest and several candidate interactors using Y2H (TakaraBio, previously clontech). If I follow manufacturer's instructions which is meant for library screening, I will need to transform separately bait into Y2HGold strain and prey into Y187.
However, in relevant publications, authors co-transformed two constructs into one yeast strain (AH109).
Selection pressures are the same between the two methods (W, L, A, H).
1). Can I test protein interactions using mated/diploid yeast rather than yeast co-transformed with 2 plasmids?
2). Are there reasons why one method is preferred over the other? Thanks.
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I always transform both bait and prey in AH109 and plate in -LT minimal media. Most of the time I will get transformants and can proceed them for higher stringency selection. However, some interactions may be toxic so in case co transformations do not work, matings can be done
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I've beeing performing different ChIP experiments with and withouth crosslinked for analysing distribution of different proteins. I've realised that when I check sonication before IP I obtain more DNA in crosslinked samples than in not-crosslinked samples. This is someting that it has also beeing observed by other collegues of my lab. Do I observe less DNA because DNA is not beeing sonicated correctly in not crosslinked samples? Should I use different sonication protocols for crosslinked and not crosslinked samples?
Right now I am using Bioruptor pico sonicator with the next protocol: 15 cycles 30s ON 30s OFF. And I am working with yeast samples.
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Mar Bustamante Sequeiros you may try adding some artificial DNA (e.g. purified PCR amplicon from qPCR reaction) to your crosslinked sample, non-crosslinked sample and handling control sample (containing no cells, but processed the same way) to see whether your PCR amplicon is preserved throughout sonication to the same extent. Drawback: using PCR amplicons together with sonicator is really messy thing ))
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Dear scientists,
I am looking for any synthetic or real RNA or DNA with a negative charge and large molecular weight (around 1M Dalton) and a reasonable price for optimization of a reaction. There is some RNA from yeast available on Sigma, but, I couldn't find the molecular weight. I would appreciate any help or suggestion.
Best regards,
Ali
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The easiest way to do this, by far, is to design primers that create a PCR product the size you're looking for (someone already calculated the number of base pairs, above) and then do large volume PCRs and purify the product. If you don't care if the nucleic acid sample is DNA or RNA, I highly highly suggest using DNA, because RNA will probably fragment under your experimental conditions and you will be handling fragments much smaller than you think you are.
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Hello, I'm trying to make media for S. cerevisiae that contains everything it needs to survive except any kind of sugar. The goal is to make stock without sugar then add in specific sugars of our choice. This is to research a gene believed to function in the metabolism of complex sugars, so we need to starve the yeast of all but specific complex sugars to test if that's actually its function.
Does anyone know how to essentially make YPD from scratch so that we have the option of leaving out any sugar containing ingredients? Thank you!
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to be 100% sure that there is no carbohydrate in the medium I would NOT USE YPD. There might be something (although in low amounts) in peptone and yeast extract. WI do recommend to use chemically defined minimal medium, such as Yeast Nitrogen Base, which is available from various suppliers.
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I have to get the telomere lenght of a few dozen yeast strains. I've read much about the standard methods but they are mostly only decribed for mammals. The problems I see are:
1. Yeast telomeres are much shorter (350 bp) and some of my mutants even shorter than WT.
2. Telomeric repeats in yeast are irregular.
Does somebody have a nicely working protocol either for qPCR (including good primers) or TRF (including good probes) that gives at least a good estimate of relative TL compared to WT?
Thanks in advance!
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Thanks Hamid! That's basically the normal TRF method. The problem is that they are quite unspecific with the method and they use radioactively labelled probes for which my lab is not accredited. I guess I'll piece together a non DIG-labelled probe and try my luck with a southern blot.
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I wanted to do the virome analysis including DNA/RNA bacteriophages and other viruses, microbiome, yeast. What is best approach? Whether we can do it in single reaction or separately, without compromizing the quality of data.
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It sounds like you want to sequence bacteria and viruses from the same sample, right? You can of course do a metagenome in one go, but you will miss some of the relevant taxa for viruses (for example RNA viruses). Also some taxa will be higher represented in the data than others due to their abundance, so you will miss the lower abundant viruses that will get less read coverage.
It depends a bit on the sample and question that you are asking. Generally I would say, that if you can separate your sample into a bacteria fraction and virus fraction, it will give you best data.
For example, some use ultracentrifugation with CsCl gradients. You would take the fraction with your intact virus capsids and do DNA and RNA extractions from it. Sequence these along with your DNA extraction of the bacteria fraction. Depending on the extraction protocol, you might introduce biases towards ssDNA, dsDNA, ssRNA and dsRNA viruses. So you probably want to have a good look into it.
Check out this paper here as a start:
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I am looking for an HA-tag antibody to use with yeast. The HA-tagged proteins I am trying to assay via WB are endogenously tagged with 3xHA. I don't see very high levels of expression. Therefore, I need a highly specific antibody with essentially no non-specific activity against yeast whole-cell extracts. Does anyone have a recommendation?
I've used a couple of antibodies 1) HA Tag Monoclonal Antibody (2-2.2.14) [Invitrogen 26183], shown below, and 2) Anti-HA (3F10) [Roche 12013819001]. Both have a lot of non-specific activity with non-tagged proteins that can be observed in the WT lane and both antibodies have the exact same background band pattern and intensity.
The calculated molecular weight is 97 kDa for SCH9-3HA and 81 kDa for YPK1-3HA.
Thank you for your suggestions!
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I've been working with HA Tag Monoclonal Antibody (2-2.2.14) [Invitrogen 26183]. I was able to get slightly less background using TBS-T with 0.1% Tween rather than 0.05 %. I used a 1:5000 dilution.
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Im working on a project and need to make sure this rough draft of a protocol is valid. Is this a good outline, am I missing any steps?
Im basing this protocol off this paper:
A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, Tong GJ, Garber M, Nnadi O, Zhuang W, Hillson NJ, Keasling JD, Mukhopadhyay A. Nucleic Acids Research. 2016 November 28; DOI: 10.1093/nar/gkw1023. PubMed PMID: 27899650.
  • Acquire dna and crispr plasmid through 3rd party supplier
  • purify dna
  • amplify dna
  • co transform yeast with dna, cas9 crispr plasmid and transformation mix
  • incubate yeast
  • examine yeast to confirm transformation
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Hi Daniel,
I would highly recommend you not to follow that paper because the authors would not provide *any* help, feedback if you face any problem.
For chromosome homology, 1000bp that was used in that paper is absolutely overkill. Don't bother yourself. Even 300-400 bp homology works well.
If you want a clear transformation protocol let me know and I will send you. However, I would strongly suggest to use this protocol:
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I work on yeast that produces oil, I want a simple and direct test to do to know if yeast produce oil or not after isolation for further studies.
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Dear Samah,
You can stain yeast cells with a dye called "Nile red". This dye is used to visualize lipid droplets and quantify neutral lipids in microorganisms, in particular the oleaginous ones. Please check these couple of references for further info and ideas.
Best regards,
Alfredo
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Hello everyone,
I want to isolate yeasts from soil samples, what is the best method to differentiate between different species of yeasts as an initial step (simple method) before molecular methods?
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For yeastlike fungi of clinical significance - try the relevant API strip. For environmental isolates, you'll need a lot more and substantial experience.
You could send isolates to folks like Accugenix for genetic based id - but that's expensive.
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I use S. cerevisiae for yeast display and I suspect I have a contamination in my cultures but it is not obvious and I don't currently have a microscope in the lab.
I grow in minimal media selecting for Trp auxotrophy complementation in the transformed yeast.
Recently my yeast cultures don't seem to display and they are growing slowly and have a less pungent smell compared to S. cerevisiae. They don't smell like E. coli either and colonies on plates have same colour and shape as S. cerevisiae as far as I can tell.
What are the common contaminants yeast scientists see and how to deal with them?
I know about Candida... is microscope the only way to identify a contamination from Candida or other species?
Thank you
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Hi Pietro,
Without a microscope or specific media culture is hard to know what is your contamination. It could be another yeast or a bacteria.
As Dominique Liger said, it would be easier to restart from your yeast stock. Make sure your new culture media is sterilized!
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Hi colleagues,
I'm trying do find the nitrogen and carbon content in yeast power or even the C/N ratio in it to adjust the C/N ration in a growing media.
Thank you!
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Yeast is one of the potential microorganisms to be utilized as a feed supplement for animals due to its high nutritional value, mainly protein and organic matter content. It could be used for varieties of substrates of agricultural and industrial by products. Yeast utilization into animal diet would improve the intestinal environment, especially on digestibility tract; prevent colonization of pathogenic bacteria in small intestine, and also systemic immune functions.The optimum C/N ratio of the fermentation medium was found to be 35.2 which gave a maximum ethanol concentration of 8.9 g/l. The optimum conditions for Co- Culture fermentation of tapioca flour were found to be the temperature 30°C, the initial pH 5.5 and the substrate concentration 60 g/l starch level using Response Surface Methodology. The maximum ethanol yield of 8.9 g/l was obtained at the optimum conditions of SSF. The logistic model and Leudeking – Piret model were found to represent closely the experimental data of growth kinetics and product formation kinetics respectively. The kinetic parameters of the models were evaluated.
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I'm looking at the impact of non-lethal levels of canavanine on some yeast strains, and I have been consistently seeing growth at the highest dilutions of serially-diluted yeast in water. I washed the yeast with water prior to diluting and subsequently spotting 5 µL on the plate. (Please see image attached.)
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Dominique Liger Thank you for your reply. I thoroughly expected and was hoping to see the growth defects of the top two strains since they should be sensitive to canavanine at the level I chose. What I don't understand is why the more diluted spots somewhat robustly recovered growth. At a slightly higher canavanine level, I do not see this dilution-recovery effect.
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We transformed yeast with lacZ and the experiment worked. We cryopreserved the yeast and now I'm not getting any beta-gal activity.
I didn't add the DTT to the buffer with the ONPG, but did add DTT to the buffer used to lyse the cells. Everything else was followed by the book.
Could this affect the results that much? Or did my yeast lose their plasmid?
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Hi there,
As mentioned above, you can't state on the assay (DTT requirement) unless you have run this assay with a positive control.
BTW why do you suspect a plasmid loss?
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I wish to know if this latest technology could be employed for heterologous expression of transporter genes in yeast.
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Please check my paper on transport engineering