Tetraploid cells grown on PRE-SPO solid medium for 48 h were collected and spheroplasted as previously described75 (link). In briefly, 1 mL of cells (OD = 2) were fixed in 4% formaldehyde for 1 h, followed by quenching with 125 mm glycine for 10 min, and then spheroplasted using 5 U of zymolyase according to the manufacturer’s instructions (G-Biosciences, St. Louis, MO). Spheroplasting was assessed visually with ~ 90% of the cells appearing as ‘ghosts’. Spheroplasted cells were then incubated with TUNEL reagents according to the manufacturer’s instructions (Roche, Basel, Switzerland) to mark DNA double-strand breaks. Cells were visualized by microscopy for fluorescence indicating DNA breaks, and an unlabeled sample served as a negative control.
Zymolyase
Manufactured by G Biosciences
Zymolyase is a non-specific yeast-lytic enzyme preparation. It contains a mixture of enzymes, including β-1,3-glucanase, that hydrolyze the cell wall of various yeast species.
Automatically generated - may contain errors
Lab products found in correlation
Acid washed glass beads, by Merck Group (2 mentions)
Tunel reagent, by Roche (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Phenol, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Mutanolysin, by Merck Group (1 mentions)
Isoamyl alcohol, by Merck Group (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Lyticase, by Merck Group (1 mentions)
Instaclone pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Plasmid miniprep kit, by Qiagen (1 mentions)
Plasmid miniprep kit, by Merck Group (1 mentions)
Ribopure yeast kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using zymolyase
1
Spheroplasting and TUNEL Assay for DNA Breaks
2
Whole-Community DNA Extraction from Cheese
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-community DNA extraction of the cheese samples was performed as described previously (Vermote et al., 2018 (link)). Briefly, the cell pellets obtained as mentioned above were submitted to an enzymatic digestion [one incubation step with lyticase (Sigma-Aldrich) and Zymolyase (G-Biosciences, St. Louis, MO, United States) and another one with lysozyme (Merck, Darmstadt, Germany) and mutanolysin (Sigma-Aldrich)], a chemical/enzymatic treatment [with sodium dodecyl sulfate (Sigma-Aldrich) and proteinase K (Merck)], and a mechanical disruption by vortexing in the presence of acid-washed glass beads (Sigma-Aldrich), followed by protein removal using a mixture of chloroform, phenol and isoamyl alcohol (Sigma-Aldrich), a treatment with RNase (Roche, Basel, Switzerland), and DNA purification with a DNeasy Blood & Tissue Kit (Qiagen, Venlo, Netherlands). The DNA purity was assessed using a NanoDrop 2000 spectrophotometer and the DNA concentrations were measured with a Qubit 2.0 fluorometer using a Qubit dsDNA HS Assay Kit (all from Thermo Fisher Scientific, Carlsbad, CA, United States).
3
Isolation and Identification of Yeast Plasmid Suppressors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Yeast cells harvested from 25 ml saturated culture of sorted libraries were resuspended in buffer P1 (supplied with Qiagen plasmid miniprep kit) and vortexed in the presence of acid-washed glass beads (SIGMA) for 10 min. The suspension was incubated with Zymolyase (30U, G-Biosciences) at 37oC for 4 hr to break the cell wall. A Qiagen plasmid miniprep kit was used for further downstream processing of the cells to purify the plasmid. CcdB gene inserts amplified from the libraries were cloned into the pTZ57R/T TA vector using an InsTAclone PCR cloning kit (ThermoScientific) and transformed into E. coli XL1-Blue cells for blue-white screening (Langley et al., 1975 (link)). The CcdB inserts from 96 randomly picked white colonies derived from each library after the final round of sorting, were sequenced by Sanger sequencing at Macrogen, Korea, to identify second-site suppressors.
4
RNA Sequencing of Dormant Fungal Spores
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We collected un‐germinated spores by first collecting 1 ml samples of spores that were incubated in a 0.002%‐glucose at 0, 16, 48 and 96 h after the incubation began. To isolate the un‐germinated spores, we treated the 1‐ml samples with zymolyase (786‐036, G‐Biosciences). zymolyase lysed vegetative cells that formed from germinated spores, thus ensuring that we only collected RNA from un‐germinated spores for sequencing. We then extracted the RNA from the leftover, un‐germinated spores RiboPure Yeast Kit (Ambion, Life Technologies) as described by its protocol. Next, we prepared the cDNA library with the 3' mRNA‐seq library preparation kit (Quant‐Seq, Lexogen) as described by its protocol. Afterwards, we loaded the cDNA library on an Illumina MiSeq with the MiSeq Reagent Kit c2 (Illumina) as described by its protocol. We analysed the resulting RNA‐seq data as previously described (Trapnell et al, 2012 ): We performed the read alignment with TopHat, read assembly with Cufflinks and analyses of differential gene expressions with Cuffdiff. We used the reference genome for S. cerevisiae from ensembl. We used the transcriptional modules listed in Appendix Table S1 for grouping the relevant genes into transcriptional modules.
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!