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.
Questions related to Yeasts
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.
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
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?
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.
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?.
Incubation period - 48-72 hrs at 30•c in a candle jar.
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!
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.
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.).
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?
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?
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?
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
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.
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 🙂.
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.
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.
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.
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)
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?
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!
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.
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?
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?
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.
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.
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
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.
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.
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.
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!
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?
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
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
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?
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?
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!
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?
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?
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.
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?
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.
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!!
For bioethanol production brewers yeast is used. But which commercial yeast is preferred for higher productivity?
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.
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.
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 ; )
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.
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?
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.
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!
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?
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.
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.
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.
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!
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!
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.
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!
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
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.
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?
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?
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.)
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?