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).
Glusulase
Manufactured by PerkinElmer
Sourced in United States
Glusulase is a laboratory reagent used for the enzymatic hydrolysis of glucosides. It contains a mixture of β-glucosidase and aryl-sulfatase enzymes. Glusulase is used to facilitate the breakdown of glucoside compounds in analytical and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Zymolase, by MP Biomedicals (4 mentions)
Coulter counter, by Beckman Coulter (2 mentions)
Fitc conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (2 mentions)
Zymolyase, by PerkinElmer (2 mentions)
Zymolyase, by ICN Biomedicals (1 mentions)
Anti rat fitc, by Jackson ImmunoResearch (1 mentions)
Anti mouse cy3, by Jackson ImmunoResearch (1 mentions)
Las4000, by GE Healthcare (1 mentions)
10 protocols using glusulase
1
Isolation and Purification of Yeast Nuclei
2
Haploid Yeast Spore Viability and Recombination
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Mating and sporulation of haploid cells were conducted as previously described with minor modifications (48 (link)). After a two-days incubation period on SPA at 30°C, cells were digested in 0.5% glusulase (PerkinElmer, Waltham, MA, USA) for 3 h to isolate spores. A total of 1000 spores were inoculated on YEA plates and grown at 30°C for 3 days, and spore viability was estimated by calculating (number of colonies grown on YEA)/1000. To measure ade6-M26 x ade6-469 intra-genic recombination frequency, 1 × 104 spores were inoculated on YEA plates followed by incubation at 30°C for 2 days so that actual numbers of viable colony were [spore viability × 10 000], then replica-plated on EMM2 without adenine (EMM2-A). The intra-genic recombination frequency of ade6 was calculated as (number of colonies grown on EMM2-A)/(spore viability × 10 000).
3
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).
4
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).
5
Microscopic Visualization of Fixed Cells
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Cells were fixed in the growth medium with 3.7% formaldehyde at room temperature for 30 min and their cell walls digested with a solution containing 50 μg/ml zymolyase and 1:50 glusulase (Perkin Elmer). The digested cells were mounted on poly-L lysine coated slides to immobilize them for microscopy. Slide preparation for all samples was finished by adding mounting medium containing DAPI to stain the DNA.
6
Mating Assay for Fission Yeast
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Mating assays were performed by mixing YSM1232 (h+), YSM3160 (h+), and YSM952 (h−; see Table S1) at different ratios. Cell mixes were plated on MSL-N plates for 24 h at 25°C. The cells were then transferred into an Eppendorf tube and treated with glusulase (1:200 from stock solution; PerkinElmer) in H2O overnight at 25°C. Only spores survived the treatment as confirmed by microscopic inspection. Spores were then plated on YE and grown for 3 d at 30°C. The plates were then replicated on selective media in order to determine the genotype of each colony.
7
Yeast Spore Viability Assay
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Spore viability was performed as previously described by Le et al. (2013 (link)) with minor modifications. Strains were mated on SPA for 48–72 hr. Asci were treated with 1:500 glusulase (Perkin Elmer, Waltham, MA) and incubated at room temperature overnight. Spore count was determined by a hemocytometer and 2000, 5000, or 10,000 spores of wild type, rec12Δ, or mutant, respectively, were plated over 10 YES plates. Spore viability was determined as the number of colonies formed over the number of plated spores. Spore viability was normalized to levels observed for the wild type.
8
Fixation and Immunostaining of Mitotic and Meiotic Cells
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Cells were fixed in growth medium with 3.7% formaldehyde at room temperature for 30 minutes. Mitotic cells were digested in SCE (1 M sorbitol/0.1 M sodium citrate/10 mM EDTA) with 20 μg/ml zymolyase (100T, ICN Biomedicals). Meiotic cells were digested with a solution containing 50 μg/ml zymolyase and 1:50 glusulase (Perkin Elmer). Cells were mounted on poly-L lysine coated slides and proteins detected by indirect immunofluorescence.
Immunostaining of whole cells was as follows: Cdc14-Myc7 fusion protein was detected with mouse monoclonal anti-Myc 9E10 antibody (Covance) at 1:600 in PBS/1% BSA for 3 hours at room temperature, followed by anti-mouse CY3 (Jackson ImmunoResearch) secondary antibody at 1:600 for 1 hour at room temperature. Slide preparation was finished by adding mounting medium containing DAPI. Detection of tubulin was done with the rat monoclonal antibody YOL 1/34, followed by anti-rat FITC (Jackson ImmunoResearch).
Immunostaining of whole cells was as follows: Cdc14-Myc7 fusion protein was detected with mouse monoclonal anti-Myc 9E10 antibody (Covance) at 1:600 in PBS/1% BSA for 3 hours at room temperature, followed by anti-mouse CY3 (Jackson ImmunoResearch) secondary antibody at 1:600 for 1 hour at room temperature. Slide preparation was finished by adding mounting medium containing DAPI. Detection of tubulin was done with the rat monoclonal antibody YOL 1/34, followed by anti-rat FITC (Jackson ImmunoResearch).
9
Intergenic and Intragenic Recombination Assay
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For intergenic recombination, haploid strains containing appropriate markers were mated and sporulated at 26.5 C on SSA plates for 30 hr. Spores were isolated using the treatment with glusulase (Perkin-Elmer) and plated on YE. The colonies were then replicated to selective media (ura1-lys3 and cnt1< 120) were examined for leucine auxotrophy, and each colony was mixed with h + and h À tester strains to determine the mating type. For intragenic recombination, h 90 haploid strains containing the ade6-M26 allele and plasmids harboring the ade6-469 allele according to the ura4 + marker were sporulated at 26.5 C on SPA plates for 24 hr. The spores were isolated by treatment with glusulase and plated on YE-containing phloxine B (to count all viable haploid spores) and SD-adenine plates. The frequency of recombination was quantified based on three independent experiments.
Detection of DNA Breaks Cells ($2 3 10 8 ) induced for meiosis were converted to spheroplasts after embedding in low-melting agarose and analyzed for PFGE, as previously described (Cervantes et al., 2000) . After the PFGE analysis, DNA was stained with Cyber Green (Takara) and detected using LAS4000 (GE).
Detection of DNA Breaks Cells ($2 3 10 8 ) induced for meiosis were converted to spheroplasts after embedding in low-melting agarose and analyzed for PFGE, as previously described (Cervantes et al., 2000) . After the PFGE analysis, DNA was stained with Cyber Green (Takara) and detected using LAS4000 (GE).
10
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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