Diploid cells were grown on YEPD plates in patches overnight and scraped off from the plates into sporulation solution [0.75% potassium acetate (Sigma)] for 2–3 days. To prepare for dissection, an aliquot of 15 μL from the sporulation culture was incubated with 15 μL of 2 mg/mL Zymolyase (Seikagaku Corporation) for 30 min at 30° to digest the spore ascus wall. Treated tetrads were dissected to individual spores using a micromanipulator microscope (Singer Instruments, Somerset, UK) on YEPD plates.
Zymolyase
Manufactured by Seikagaku
Sourced in Japan
Zymolyase is a commercial enzyme preparation derived from the culture filtrates of Arthrobacter luteus. It primarily contains β-1,3-glucanase and protease activities, which are effective for the digestion of fungal cell walls. The product is commonly used in the isolation and preparation of fungal protoplasts.
Automatically generated - may contain errors
Lab products found in correlation
Potassium acetate, by Merck Group (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Anion exchange resin, by Qiagen (1 mentions)
Nextflex rapid dna seq kit, by PSC Biotech (1 mentions)
Genomic tip 20 g kit, by Qiagen (1 mentions)
S2 ultrasonicator, by Covaris (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Mnase, by Takara Bio (1 mentions)
Chef mapper, by Bio-Rad (1 mentions)
Sybr green, by Lonza (1 mentions)
Lipopolysaccharides lps from escherichia coli o111 b4, by Merck Group (1 mentions)
Masterpure yeast dna purification kit, by Illumina (1 mentions)
Dnase 1, by Promega (1 mentions)
Genejet rna cleanup and concentration micro kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 4000 sequencing technology, by Illumina (1 mentions)
Prestained protein standards, by Bio-Rad (1 mentions)
Streptavidin magnetic beads, by Promega (1 mentions)
Oligonucleotide, by Integrated DNA Technologies (1 mentions)
Sodium ascorbate, by Merck Group (1 mentions)
10 protocols using zymolyase
1
Sporulation and Tetrad Dissection Protocol
2
Yeast Chromatin Fractionation and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yeast chromatin was fractionated as previously described (Keogh et al., 2005 (link)) with modifications. 40 to 50 OD cells were collected and washed with SB buffer (1M Sorbitol, 20 mM Tris pH 7.5). Pellets were successively washed with PSB (20 mM Tris pH 7.5, 100 mM NaCl, 20 mM EDTA, 10 mM β-Mercaptoethanol), SB buffer then digested with 1 mg/mL Zymolyase (SEIKAGAKU) in SB buffer for 1 hr at RT. Spheroplasts were washed with SB, then lysed in ice by 0.5% EBX (20 mM Tris pH 7.5, 100 mM NaCl, 15% β-Mercaptoethanol, 0.5% Triton X-100). Lysates with 0.5% EBX were layered by NIB buffer (20 mM Tris pH 7.5, 100 mM NaCl, 15% β-Mercaptoethanol, 1.2M Sucrose), then fractionated by centrifugation at 12,000 rpm, 4°C for 15 min. Pellets were resuspended and lysed again in 1% EBX (20 mM Tris pH 7.5, 100 mM NaCl, 15% β-Mercaptoethanol, 1% Triton X-100). Lysates were fractionated by centrifugation at 14,000 rpm, 4°C for 10 min. Pellets were resuspended with 2X sample buffer and boiled at 100°C for 5 min.
3
Comprehensive Genomic and Transcriptomic Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For genomic sequencing, CLIB89 was cultured in 2% Yeast extract-1% Peptone 2%-Dextrose (YPD) [63 ]. For Illumina sequencing DNA was extracted according to standard protocols; RNA was removed by RNase digestion [64 ]. DNA was sheared to appropriate length using the S2 Ultrasonicator (Covaris). Libraries were prepared for sequencing using the NEXTflex Rapid DNA-Seq Kit (Bioo Scientific). For PacBio sequencing, cells were spheroplasted by treatment with Zymolyase (Seikagaku Corporation). Spheroplasts were pelleted, lysed and digested with RNaseA (Fermentas) and Proteinase K (Fisher Scientific). DNA was isolated and then eluted from the Qiagen Anion-Exchange Resin. For datasets YLP13 and YLP14, corresponding to PacBio RS sequencing, high MW DNA was extracted with the Genomic-Tip 20/G kit (Qiagen). DNA was fractionated to 4–50 kb using a BluePippin pulsed-field gel electrophoresis system (Sage Sciences). PacBio SMRT Bell sequencing libraries were prepared using the manufacturer’s DNA SMRT kit. Results of RNA sequencing will be published elsewhere. Transcripts from cells grown under several conditions were combined in order to maximize the potential of transcriptomics to identify reading frames. For RNA sequencing cells were lysed, and RNA was processed into KAPA stranded libraries for Illumina PE100 sequencing according to manufacturer’s instructions.
4
Isolation and Characterization of Yeast Mitochondria
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondria were isolated by differential centrifugation. Yeast cells were grown to an OD600 of 1 in YPG or YPS medium. Cells were harvested by centrifugation at 2,500 g, washed with distilled H2O, and incubated in 2 ml of 0.1-M Tris, pH 9.4, and 10-mM DTT per gram wet weight of cells for 20 min at 24°C (Meisinger et al., 2006 (link)). Cells were reisolated and washed with Zymolyase buffer (1.2-M sorbitol and 20-mM K2HPO4, pH 7.4). To digest the cell wall, cells were incubated with 4 mg Zymolyase (Seikagaku) per gram cell pellet in 7 ml per gram cell pellet Zymolyase buffer for 40 min at 24°C. After a further washing step with Zymolyase buffer, the spheroblasts were homogenized on ice in homogenization buffer (0.6-M sorbitol, 10-mM Tris, pH 7.4, 1-mM EDTA, 1-mM PMSF, and 0.2% [wt/vol] BSA) with a glass potter. Cell debris was removed by centrifugation at 2,500 g, and mitochondria were pelleted at 17,000 g. The mitochondrial pellet was washed with SEM buffer (250-mM sucrose, 1-mM EDTA, and 10-mM MOPS/KOH, pH 7.2) and resuspended in SEM buffer. The protein concentration was adjusted to 10 mg/ml. The mitochondria were shock frozen in liquid nitrogen and stored at −80°C.
5
Isolation and Purification of Mononucleosomal DNA from S. complicata
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal volumes of S. complicata culture and 2% formaldehyde were mixed and incubated for 10 min. Next, 5 mL of 1.25 M glycine was added to the resulting solution. S. complicata cells were collected, washed with 50 mM Tris-EDTA buffer (pH 8), and then suspended in Zymolyase buffer (1 M sorbitol, 10 mM DTT, and 50 mM Tris-HCl, pH 8.0). Zymolyase (Seikagaku corporation, Japan) (50 U) was added to the cell suspension, and the resulting solution was incubated at 37 °C for 1 h. Cells were collected by centrifugation and suspended in 2.5 mL of Zymolyase buffer, after which 1 U of MNase (Takara, Japan) was added. The resulting digestion solution was incubated at 37 °C for 30 min, and the reaction was stopped by adding sodium dodecyl sulfate to a final concentration of 1% and EDTA to a final concentration of 10 mM. Proteinase K solution (5 µL) was added to the solution, and the mixture was incubated at 56 °C for 1 h. DNA was phenol/chloroform-extracted, ethanol-precipitated, and treated with RNase (Nippon Gene, Japan). Nucleosomal DNA fragments were isolated via electrophoresis on 2% agarose gel. The mononucleosomal DNA band was excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Germany).
6
Pulsed-Field Gel Electrophoresis of Chromosomal DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromosomal DNA embedded in 1% InCert agarose plugs (Lonza, Basel, Switzerland) were treated using zymolyase (Seikagakukogyo), RNase A, and proteinase K. Plugs were electrophoresed on a 1% Magabase agarose (Bio-Rad, Hercules, CA, USA) gel in 0.5× Tris/borate/EDTA buffer using CHEF Mapper (Bio-Rad). Electrophoresis was performed out at 14 °C with 6 V/cm voltage for 24 h. The angle was 120°, and the pulse time was ranged from 60 to 120 s. The DNA was stained with SYBR Green (Lonza, Rockland, ME, USA).
7
Fungal Strain Cultivation and Reagent Sourcing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All strains of A. fumigatus (NBRC 30870 and 4400), A. niger (NBRC 6342), Aspergillus oryzae (NBRC 30103), and Candida albicans (NBRC 1385) were purchased from the NITE Biological Resource Center (Chiba, Japan), maintained on Sabouraud agar (Difco, Detroit, MI, USA) at 25℃, and transferred once every 3 months. Fungitec G test MK was purchased from Nissui Pharmaceutical Co. (Tokyo, Japan). Zymolyase was obtained from Seikagaku Corp (Tokyo, Japan). Lipopolysaccharides (LPS) from Escherichia coli O111: B4 were purchased from Sigma (St. Louis, MO, USA). Dextran was purchased from Seikagaku Corp. Polyglobin N was purchased from Bayer AG, Pharmaceuticals (Berlin, Germany). Kenketu glovenin-Ⅰ was purchased from Nihon Pharmaceutical Co., Ltd. GAMMAGARD was purchased from Baxalta Japan, Ltd. (Tokyo, Japan). Human reference serum was purchased from Sigma and Bethyl Laboratories (TX, USA).
8
Genomic DNA and RNA Extraction from Yeast
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from T0, T1, and T2 sampling time points of the selection experiments. Cultures were harvested by centrifugation and cells were treated with 10 units of Zymolyase (Seikagaku Corporation, Japan) for 30 min at 37°C. Then, genomic DNA was extracted using the MasterPure Yeast DNA Purification Kit (Epicentre-Illumina, United States) according to the supplier’s instructions. Total RNA was extracted from the T2 time point of selection experiment using the E.Z.N.A. Total RNA Kit I (OMEGA, United States) according to the supplier’s instructions. The RNA solution was treated with DNase I (Promega) to remove genomic DNA traces and recovered using the GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Scientific, United States). Purified DNA and RNA concentrations were determined using an UV-Vis spectrophotometer EPOCH equipment (Biotek, United States) and verified by 1.5% agarose gels. Finally, DNA and RNA sequencing (including library preparation) were performed by Illumina HiSeq 4000 sequencing technology, obtaining paired-end read of 100 bp through the service of the Beijing Genomic Institute (BGI, China).
9
Yeast Protein Extraction and Analysis
10
Purification of Yeast Mitochondria
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!