DMEM low glucose media, DMEM high glucose media, Hams’ F‐12 media, DMEM/F‐12 media (1:1), penicillin/streptomycin solution and phosphate buffered saline (PBS) were purchased from Invitrogen (Carlsbad, CA). The media additives d‐biotin, adenine hemisulfate, insulin solution, apo‐transferrin, and Nuclei EZ Prep kit were purchased from Sigma–Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). Charcoal stripped FBS (CSS) was prepared within our laboratory or purchased from Invitrogen (Carlsbad, CA). Zapoglobin and Isoton II were purchased from Beckman Coulter Inc. (Fullerton, CA). Rabbit anti‐mouse IgG secondary antibody was obtained from Zymed Laboratories, Inc. Both horseradish peroxidase‐conjugated donkey anti‐rabbit and sheep anti‐mouse antibodies were purchased from GE Healthcare Biosciences (Pittsburg, PA). All tissue culture plasticware and additional chemicals were purchased from Fisher Scientific (Suwanee, GA).
Adenine hemisulfate
Manufactured by Merck Group
Sourced in United States
Adenine hemisulfate is a chemical compound used in various laboratory applications. It serves as a building block for the synthesis of nucleic acids, such as DNA and RNA, and plays a role in cellular energy metabolism. Adenine hemisulfate is a white crystalline powder that is soluble in water and other polar solvents.
Automatically generated - may contain errors
Lab products found in correlation
Uracil, by Merck Group (5 mentions)
L tryptophan, by Merck Group (4 mentions)
L histidine, by Merck Group (4 mentions)
Yeast nitrogen base without amino acids, by BD (3 mentions)
Dextrose, by Thermo Fisher Scientific (3 mentions)
L phenylalanine, by Merck Group (3 mentions)
L methionine, by Merck Group (3 mentions)
L threonine, by Merck Group (3 mentions)
L valine, by Merck Group (3 mentions)
L isoleucine, by Merck Group (3 mentions)
L tyrosine, by Merck Group (3 mentions)
L arginine, by Merck Group (3 mentions)
L lysine, by Merck Group (3 mentions)
L leucine, by Merck Group (3 mentions)
Bacto peptone, by BD (3 mentions)
Insulin solution, by Merck Group (2 mentions)
Zapoglobin, by Beckman Coulter (2 mentions)
Isoton 2, by Beckman Coulter (2 mentions)
Horseradish peroxidase conjugated donkey anti rabbit and sheep anti mouse antibodies, by GE Healthcare (2 mentions)
Apo transferrin, by Merck Group (2 mentions)
19 protocols using adenine hemisulfate
1
Cell Culture Media and Reagents Procurement
2
Cultivation of Candida albicans Strains
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Cultures of C. albicans serotype A strain CAI4+CIp10 (NGY152) and the Δefg1/Δefg1 strain (CA79) obtained from glycerol stocks frozen at −80°C were maintained on solid synthetic complete medium without uracil (SC-Ura medium) prepared from 0.67% (wt/vol) yeast nitrogen base without amino acids (Formedium, Norfolk, United Kingdom), 2% (wt/vol) technical agar (Oxoid, Cambridge, United Kingdom), 1 mM NaOH solution (BDH Chemicals, VWR International, Leicestershire, United Kingdom), double-distilled H2O, 0.1% (wt/vol) adenine hemisulfate (Sigma-Aldrich, Dorset, United Kingdom), 4% (wt/vol) glucose (Fisher Scientific, Leicestershire, United Kingdom), and 0.4% SC-Ura dropout mixture (Formedium). A single colony of C. albicans from the plate was cultured in 5 ml of SC-Ura liquid medium (the same recipe as above, without the agar) at 30°C overnight with shaking at 200 rpm.
3
Nonselective Yeast Growth Media
4
Yeast Strain Manipulation Protocol
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The parental wild-type yeast strain used in this study was BY4741. All gene deletions, genomic integrations, and plasmid transformations were done in this background using standard methods. For a complete list of strains used, see S1 Table .
Yeast cultures were grown at 30°C in YPD or synthetic defined (SD) media with appropriate nutrient dropouts. SD media used for growth of yeast cultures contained: 2% w/v dextrose (Thermo Fisher Scientific, Waltham, MA), 13.4 g/L Yeast Nitrogen Base without Amino Acids (BD Biosciences, San Jose, CA), 0.03 g/L L-isoleucine (Sigma-Aldrich, St. Louis, MO), 0.15 g/L L-valine (Sigma-Aldrich), 0.04 g/L adenine hemisulfate (Sigma-Aldrich), 0.02 g/L L-arginine (Sigma-Aldrich), 0.03 g/L L-lysine (Sigma-Aldrich), 0.05 g/L L-phenylalanine (Sigma-Aldrich), 0.2 g/L L-Threonine (Sigma-Aldrich), 0.03 g/L L-tyrosine (Sigma-Aldrich), 0.018 g/L L-histidine (Sigma-Aldrich), 0.09 g/L L-leucine (Sigma-Aldrich), 0.018 g/L L-methionine (Sigma-Aldrich), 0.036 g/L L-tryptophan (Sigma-Aldrich), and 0.018 g/L uracil (Sigma-Aldrich). Media additionally contained 5 mM or 10 mM guanidinium hydrochloride (Sigma-Aldrich) where indicated and bortezomib (LC Laboratories, Woburn, MA) treatment lasted 4 hours.
Yeast cultures were grown at 30°C in YPD or synthetic defined (SD) media with appropriate nutrient dropouts. SD media used for growth of yeast cultures contained: 2% w/v dextrose (Thermo Fisher Scientific, Waltham, MA), 13.4 g/L Yeast Nitrogen Base without Amino Acids (BD Biosciences, San Jose, CA), 0.03 g/L L-isoleucine (Sigma-Aldrich, St. Louis, MO), 0.15 g/L L-valine (Sigma-Aldrich), 0.04 g/L adenine hemisulfate (Sigma-Aldrich), 0.02 g/L L-arginine (Sigma-Aldrich), 0.03 g/L L-lysine (Sigma-Aldrich), 0.05 g/L L-phenylalanine (Sigma-Aldrich), 0.2 g/L L-Threonine (Sigma-Aldrich), 0.03 g/L L-tyrosine (Sigma-Aldrich), 0.018 g/L L-histidine (Sigma-Aldrich), 0.09 g/L L-leucine (Sigma-Aldrich), 0.018 g/L L-methionine (Sigma-Aldrich), 0.036 g/L L-tryptophan (Sigma-Aldrich), and 0.018 g/L uracil (Sigma-Aldrich). Media additionally contained 5 mM or 10 mM guanidinium hydrochloride (Sigma-Aldrich) where indicated and bortezomib (LC Laboratories, Woburn, MA) treatment lasted 4 hours.
5
Benzimidazole and Purine Derivative Synthesis
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Chemicals were obtained from the sources indicated: 5′-chloro-5′-deoxyadenosine, Santa Cruz Biotechnology; 7-methylbenzimidazole, Accela; 5-methyl-1H-benzimidazole, Acros Organics; phenol, J. T. Baker; zinc metal, Fisher Scientific; 5-methoxybenzimidazole, purine, and para-cresol, Alfa Aesar; methylmalonyl-CoA, methylmalonic acid, coenzyme A, adenosylcobalamin (coenzyme B12), cyanocobalamin, dicyanocobinamide, 6-methylpurine, 1H-imidazo[4,5-c]pyridine-4-amine (3-deazaadenine), benzimidazole, adenine hemisulfate, 5-azabenzimidazole, 1H-benzo[d]imidazol-7-amine (7-aminobenzimidazole), 2-methyl-1H-purine-6-amine (2-methyladenine), and bovine serum albumin (BSA), Sigma.
6
Cultivation and Transformation of E. coli and S. cerevisiae
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E. coli strains XL-1 Blue and DH5α were cultivated in 5 mL lysogeny broth (LB) containing 150 μg/mL ampicillin for molecular cloning and plasmid maintenance. S. cerevisiae strains were grown at 30˚C in 15 × 125-mm borosilicate culture tubes containing 5 mL medium: YPD [10 g/L yeast extract (BD Difco™, BD, Franklin Lakes, NJ), 20 g/L peptone (BD Difco™), and 20 g/L D-glucose (Fisher Scientific, Hampton, NH)] prior to plasmid transformation, and selective SDC(A) [20 g/L D-glucose, 1.7 g/L yeast nitrogen base without amino acids (BD Difco™), 5 g/L casamino acids (BD Difco™), 100 mg/L adenine-hemisulfate (Sigma-Aldrich, St. Louis, MO), and 5 g/L ammonium sulfate (Fisher Scientific)] following transformation. Detailed yeast transformation methods can be found in SI Appendix, Supplemental Methods . For acetyl-CoA and TAL production, cells from overnight cultures in SDC(A) were reinoculated into SDC(A) (with 10 g/L D-glucose) and cultured for 48 or 96 h. Optical density (OD600) was measured using a Shimadzu UV‐2450 UV‐Vis spectrophotometer (Shimadzu, Columbia, MD) and converted to cell density (gDCW/L) via a linear correlation. Cells were harvested by centrifugation at 3,000 rcf in a Beckman Coulter Allegra X-22R Centrifuge (Beckman Coulter, Brea, CA).
7
Yeast Growth Curve Analysis with Cas9
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Yeast cells containing pCAS (Cas9-His8 variant) were grown in a Bioscreen C Growth Curve Analyzer (Growth Curve USA, Piscataway, NJ) in 200 µl of YPD + G418 liquid medium (20 g/l Peptone [211667; Bacto], 10 g/l Yeast Extract [212750; Bacto], 0.15 g/l Adenine hemisulfate [A9126; Sigma] and 20 g/l Glucose [G8270; Sigma] + 200 mg/l G418 [29065A; Santa Cruz Biotechnology]). Cells were grown in three biological replicates each with five technical replicates for 48 hr at 30°C under constant shaking. The wild-type control containing an empty vector (pOR1.1) was also grown in five technical replicates. Mean and standard deviations of the optical density at 600 nm were calculated for each time point measured by the Bioscreen.
8
Yeast Culture Conditions for URA3 Plasmid Assays
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Cells expressing URA3 plasmids (pDRF1-GW, pYES2, or pYX212) were grown overnight in 1x YNB medium (Sigma–Aldrich, St. Louis, MO, USA), containing 1% ethanol (Boom BV, Meppel, The Netherlands), 20 mg/L adenine hemisulfate (Sigma–Aldrich), 20 mg/L L-tryptophan (Sigma–Aldrich), 20 mg/L L-histidine (Sigma–Aldrich), and 60 mg/L L-leucine (SERVA Electrophoresis GmbH, Heidelberg, Germany). For WT strains, uracil (Sigma–Aldrich) was added to a final concentration of 20 mg/L. The cells were subsequently diluted and grown overnight to an OD600 of 0.1–1.5, with at least 5 divisions.
For the experiments that involved S25-31C, cells were grown on 1x YNB medium containing 1% ethanol, 5 mM glucose (Boom BV, Meppel, The Netherlands), 20 mg/L adenine hemisulfate, 20 mg/L L-tryptophan, 20 mg/L L-histidine, and 60 mg/L L-leucine until glucose was exhausted. Next, cells were kept on this medium for 2 more days, after which they were visualized under the microscope.
For the experiments that involved S25-31C, cells were grown on 1x YNB medium containing 1% ethanol, 5 mM glucose (Boom BV, Meppel, The Netherlands), 20 mg/L adenine hemisulfate, 20 mg/L L-tryptophan, 20 mg/L L-histidine, and 60 mg/L L-leucine until glucose was exhausted. Next, cells were kept on this medium for 2 more days, after which they were visualized under the microscope.
9
Autophagy Induction in Budding Yeast
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All experiments were performed at 30°C. Budding yeast (S. cerevisiae) were cultured in YPA (1% Bacto-yeast extract (Y), 2% Bacto-peptone (P), 0.025% adenine hemi-sulfate (A; Sigma)) supplemented with 2% glucose (D; Sigma) or in csm (complete supplement mixture; Sunrise Science Products) to maintain selection for centromeric plasmids. For one experiment, we used S. pombe strain MKSP3186, which was cultured overnight in YE5S (yeast extract (YE), 250 mg/L adenine, histidine, leucine, uracil, and lysine hydrochloride).
To induce autophagy by nitrogen starvation, cells in log phase were washed and resuspended in synthetic defined medium lacking nitrogen (sd-N) (0.17% Difco Yeast Nitrogen Base without amino acids and ammonium sulfate, and 2% D). Samples were collected at time points indicated in the figures and were prepared for extraction of RNA, fluorescence microscopy, or immunoblotting as described below.
To induce autophagy by inhibiting Tor1 kinase by treatment with rapamycin, rapamycin (in DMSO; Sigma-Aldrich) was added to cells in log phase to a final concentration of 250 ng/mL. Samples collected at time points indicated in the figures were prepared for extraction of RNA for RT-qPCR.
To induce autophagy by nitrogen starvation, cells in log phase were washed and resuspended in synthetic defined medium lacking nitrogen (sd-N) (0.17% Difco Yeast Nitrogen Base without amino acids and ammonium sulfate, and 2% D). Samples were collected at time points indicated in the figures and were prepared for extraction of RNA, fluorescence microscopy, or immunoblotting as described below.
To induce autophagy by inhibiting Tor1 kinase by treatment with rapamycin, rapamycin (in DMSO; Sigma-Aldrich) was added to cells in log phase to a final concentration of 250 ng/mL. Samples collected at time points indicated in the figures were prepared for extraction of RNA for RT-qPCR.
10
Studying Heh1 Alleles in Yeast
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All strains used in this study are from a W303 parent; their derivation and genotypes are listed in Supplementary file 1 . Fluorescent protein tagging and gene deletions were generated using a PCR-based integration approach using the pFA6a plasmid series (Supplementary file 2 ) as templates (Longtine et al., 1998 (link); Van Driessche et al., 2005 (link)). Standard yeast protocols for transformation, mating, sporulation, chromosomal DNA isolation and tetrad dissection were followed (Amberg et al., 2005 ).
Cells were grown to mid-log phase in YPA (1% Bacto yeast extract (BD), 2% Bacto peptone (BD), 0.025% adenine hemi-sulfate (Sigma)) or complete synthetic medium (CSM) supplemented with 2% raffinose (R; BD), 2% D-galactose (G; Alfa Aesar) or 2% D-glucose (D; Sigma) as indicated.
To compare relative growth rates of heh1Δapq12Δ strains expressing HEH1 alleles (DTCPL1498, DTCPL1517, DTCPL1581, DTCPL1519, DTCPL1520) roughly equivalent cell numbers from overnight cultures grown in in YPAR were spotted in 10-fold serial dilutions onto YPG to induce expression of Heh1 or indicated truncations and imaged after 36 hr at 30oC.
Cells were grown to mid-log phase in YPA (1% Bacto yeast extract (BD), 2% Bacto peptone (BD), 0.025% adenine hemi-sulfate (Sigma)) or complete synthetic medium (CSM) supplemented with 2% raffinose (R; BD), 2% D-galactose (G; Alfa Aesar) or 2% D-glucose (D; Sigma) as indicated.
To compare relative growth rates of heh1Δapq12Δ strains expressing HEH1 alleles (DTCPL1498, DTCPL1517, DTCPL1581, DTCPL1519, DTCPL1520) roughly equivalent cell numbers from overnight cultures grown in in YPAR were spotted in 10-fold serial dilutions onto YPG to induce expression of Heh1 or indicated truncations and imaged after 36 hr at 30oC.
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