Diploid of WT, lcb4Δ, lag1Δ with Nup82-GFP cells and WT with Rtn1-GFP were used for preparation of yeast nuclei. The methods are described in Schreiner etal. (2015) (link). Briefly, exponentially growing yeast cells were taken and treated with 100 mM of Tris-HCl (pH 9.4) and 10 mM DTT for 10 minutes at 25°C. After washing cells with enzyme buffer (1.1 M sorbitol, 20 mM of KCl, 0.5 mM of MgCl2, pH 7.4 with NaOH), cells were incubated with 200 μg/mL of zymolase (MP biomedicals) and 100 μL of glusulase (PerkinElmer) in enzyme buffer for 1 hour at 30°C. Spheroplasts were applied on Ficoll-sorbitol cushion (7.5% of Ficoll and 1.1 M sorbitol) and centrifuged at 10,000 × g for 15 minutes at 4°C. After harvesting spheroplasts, cells were broken using Dounce homogenizer (Wheaton, type B) in 8% Polyvinylpyrrolidone, 10 mM of Tris-HCl (pH 6.4), 0.5 mM of MgCl2 and proteinase inhibitor cocktail. Lysates were loaded on three density step sucrose gradients (1.8, 2.3 and 2.5 M) and centrifuged at 15,000 × g for 1.5 hours at 4°C. Nuclei were harvested between 1.8 and 2.3 M sucrose gradient and then the number of nuclei were counted using a Coulter Counter (Beckman).
Zymolase
Manufactured by MP Biomedicals
Zymolase is a type of enzyme that is used for the digestion of yeast cell walls. It is commonly used in molecular biology applications, such as the isolation of yeast DNA or the preparation of yeast protoplasts.
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Lab products found in correlation
6 protocols using zymolase
1
Isolation and Purification of Yeast Nuclei
2
Isolation and Purification of Yeast Nuclei
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Diploid of WT, lcb4Δ, lag1Δ with Nup82-GFP cells and WT with Rtn1-GFP were used for preparation of yeast nuclei. The methods are described in Schreiner etal. (2015) (link). Briefly, exponentially growing yeast cells were taken and treated with 100 mM of Tris-HCl (pH 9.4) and 10 mM DTT for 10 minutes at 25°C. After washing cells with enzyme buffer (1.1 M sorbitol, 20 mM of KCl, 0.5 mM of MgCl2, pH 7.4 with NaOH), cells were incubated with 200 μg/mL of zymolase (MP biomedicals) and 100 μL of glusulase (PerkinElmer) in enzyme buffer for 1 hour at 30°C. Spheroplasts were applied on Ficoll-sorbitol cushion (7.5% of Ficoll and 1.1 M sorbitol) and centrifuged at 10,000 × g for 15 minutes at 4°C. After harvesting spheroplasts, cells were broken using Dounce homogenizer (Wheaton, type B) in 8% Polyvinylpyrrolidone, 10 mM of Tris-HCl (pH 6.4), 0.5 mM of MgCl2 and proteinase inhibitor cocktail. Lysates were loaded on three density step sucrose gradients (1.8, 2.3 and 2.5 M) and centrifuged at 15,000 × g for 1.5 hours at 4°C. Nuclei were harvested between 1.8 and 2.3 M sucrose gradient and then the number of nuclei were counted using a Coulter Counter (Beckman).
3
Meiotic Staging by Fluorescence Microscopy
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Meiotic staging was performed scoring DAPI and tubulin morphology by fluorescent microscopy. Samples were fixed in 3.7% formaldehyde for 12–24 h at 4°C. Cells were then washed with 100 mM potassium phosphate, pH 6.4, once with sorbitol citrate (100 mM potassium phosphate, pH 7.5, and 1.2 M sorbitol), and digested in 200 µl sorbitol citrate, 20 µl glusulase (Perkin-Elmer), and 6 µl zymolase (10 mg/ml; MP Biomedicals) for 3 h at 30°C while rotating. Samples were pelleted at 900 rcf for 2 min, washed with 100 µl sorbitol citrate, pelleted again, and resuspended in 50 µl sorbitol citrate. Samples were then mounted on slides prepared with poly-L -lysine, submerged in 100% methanol at −20°C for 3 min, transferred to 100% acetone at −20°C for 10 s, and allowed to air dry. Samples were then incubated at RT for 1 h in primary anti-tubulin antibody (RRID:AB_325005, MCA78G, 1:200; Bio-Rad) in PBS-BSA (5 mM potassium phosphate, 15 mM NaCl, 1% BSA, and 0.1% sodium azide). Samples were then washed three times in PBS-BSA and incubated with preabsorbed FITC-conjugated secondary antibody (RRID:AB_2340652, 712–095-153, 6:200; Jackson ImmunoResearch Labs) for 1 h at RT. Samples were washed three times with PBS-BSA and mounted with VectaShield Antifade Mounting Medium with DAPI (Vector Labs).
4
Glucose Effects on Yeast Histone Modifications
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To examine the effect of glucose on histone modifications, yeast cells were grown in 2% glucose-containing YPD (Y east Extract P eptone D extrose) medium until OD600 of 0.7–1.0. Cells were then collected, washed and resuspended in YP medium for 3 hr followed by treatment with different concentrations of glucose for 0.5 hr. For 1NM-PP1 (MCE, HY-13804) treatment, cells were grown in 2% YPD until OD600 of 0.7–1.0. Cells were then treated with 25 μM 1NM-PP1 for 0.5 hr. For FBP and pyruvate treatment, cells were pre-treated with Zymolase (Mpbio, 08320921) to increase the permeability. In brief, cells were grown in YPD medium until OD600 of 0.7–1.0, centrifuged and resuspended in 1 ml SB buffer (100 mM Sorbitol, 20 mM Tris pH 7.4). 10 μl Zymolase was then added and incubated at 30 °C for 15 min. The spheroplasts were washed and cultured in YPD medium containing different concentrations of FBP and pyruvate for 3 hr.
5
Isolation of Yeast Cell Nuclei
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Nuclei were isolated using a protocol previously described in (Schreiner et al., 2015) .
Briefly, strains were grown to log phase in rich media (YE5S) at 30°C and diluted into 1 liter YE5S for overnight growth. Cells were harvested the following day at an OD600 ~0.8 and incubated in 100 mM Tris pH 9.4 and 10 mM DTT for 10 min at 33°C to prepare for spheroplasting. Cells were spheroplasted in 0.4 mg/mL zymolase (MP Biomedicals), 0.6 mg/mL lysing enzymes (Sigma), 350 𝜇L beta-glucuronidase (MP Biomedicals) and 5 mM DTT in 1.1 M sorbitol at 33°C for 2-3 hours. Spheroplasts (nuclei) were isolated from cells by centrifugation using a series of three density-step sucrose gradients, and aliquots were flash frozen in liquid N2 and stored at -80°C. To thaw for use, one aliquot was dialyzed overnight in 500 mL dialysis buffer (80 mM PIPES, 5% DMSO, 2 mM MgCl2, 1 mM EGTA, 500 mM sucrose).
Briefly, strains were grown to log phase in rich media (YE5S) at 30°C and diluted into 1 liter YE5S for overnight growth. Cells were harvested the following day at an OD600 ~0.8 and incubated in 100 mM Tris pH 9.4 and 10 mM DTT for 10 min at 33°C to prepare for spheroplasting. Cells were spheroplasted in 0.4 mg/mL zymolase (MP Biomedicals), 0.6 mg/mL lysing enzymes (Sigma), 350 𝜇L beta-glucuronidase (MP Biomedicals) and 5 mM DTT in 1.1 M sorbitol at 33°C for 2-3 hours. Spheroplasts (nuclei) were isolated from cells by centrifugation using a series of three density-step sucrose gradients, and aliquots were flash frozen in liquid N2 and stored at -80°C. To thaw for use, one aliquot was dialyzed overnight in 500 mL dialysis buffer (80 mM PIPES, 5% DMSO, 2 mM MgCl2, 1 mM EGTA, 500 mM sucrose).
6
Meiotic Staging by Fluorescence Microscopy
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Meiotic staging was performed scoring DAPI and tubulin morphology by fluorescent microscopy as described in Sawyer et. al. (2019) . Samples were fixed in 3.7% formaldehyde for 12-24 hr at 4° C. Cells were then washed with 100 mM potassium phosphate pH6.4, once with sorbitol citrate (100 mM potassium phosphate pH7.5, 1.2 M sorbitol), and digested in 200 µL sorbitol citrate, 20 µL glusulase (Perkin-Elmer) and 6 µL of zymolase (10 mg/mL, MP Biomedicals) for 3 hours at 30° C while rotating. Samples were pelleted at 900 rcf for 2 min, washed with 100 µL sorbitol citrate, pelleted again and resuspended in 50 µL sorbitol citrate. Samples were then mounted on slides prepared with poly-L-lysine, submerged in 100% methanol at -20° C for 3 min, transferred to 100% acetone at -20° C for 10 sec, then allowed to air dry. Samples were then incubated at RT for 1 hr in primary anti-tubulin antibody (Bio-Rad, 1:200) in PBS-BSA (5 mM potassium phosphate, 15 mM NaCl, 1% BSA, 0.1% sodium azide). Samples were then washed 3x in PBS-BSA and incubated with preabsorbed FITC-conjugated secondary antibody (Jackson ImmunoResearch Labs, 6:200) for 1 hr at RT. Samples were washed 3x with PBS-BSA and mounted with VectaShield Antifade Mounting Medium with DAPI (Vector Labs).
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