NEEDS REPHRASING:
Yeast cells were grown in 3 mL of YPD broth at 30°C, 170 rpm for three days and then pelleted and washed with sterile ddH2O. The cells were resuspended in 200µL of breaking buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl). 0.3 g of glass beads and 200µL of phenol/chloroform/isoamyl alcohol was added and the samples were vortexed at maximum speed for 3 mins in order to lyse the cells. Following centrifugation, the aqueous layer was transferred to a clean tube and 1 mL of 100% ethanol was added. The supernatant was removed following another centrifugation step. The resulting pellet was resuspended in 400µL of 1X TE buffer and 30µL of 1 mg/mL DNase-free RNase A and incubated at 37°C for 5 mins. The pellet was then washed with 1 mL of 100% ethanol and 10µL of 4M ammonium acetate, followed by another wash with 1 mL of 70% ethanol, and then resuspended in 100µL of sterile ddH2O and frozen at -20ºC for storage.
Could also just reference protocol in “Short protocols in molecular biology”
PCR and Sequencing
Needs Rephrasing
The internally transcribed spacer (ITS) regions of the Kveik yeast strains were amplified using ITS1 and ITS4 primers (Pham et al., 2011). PCR reactions contained 1µL of genomic DNA, 2.5µM of each primer, 0.4 mM dNTPs, 2.5 U of Taq DNA polymerase, and 1X Taq reaction buffer. The amplification reactions were carried out in a BioRad T100 Thermal cycler under conditions adapted from Pham et al., 2011.
PCR products were visualized on a 1% agarose gel in 1X TAE buffer to ensure successful amplification. The samples were purified using a QIAquick PCR purification kit and submitted for sequencing using an Applied Biosystems 3730 DNA analyzer. What about quality control (4peaks)? The resulting sequences were identified using NCBI BLAST.
PCR of ITS1/ITS4
Sequencer used
DNA Fingerprinting
Need the pcr specifics and primers
Reaction products were confirmed through electrophoresis on a 1% agarose gel in 1X TAE buffer. PCR samples were then purified using a QIAquick PCR purification kit and submitted for analysis on an Agilent 2100 Bioanalyzer using the Agilent DNA 7500 chip.
Banding patterns obtained using Bioanalyzer were analyzed using GelJ software and a dendrogram was built using the software, using Pearson correlation and the unweighted pair group method with arithmetic mean (UPGMA) cluster algorithm (Heras et al. 2015).
Wort Preparation
Wort used for beer fermentations and yeast propagation was obtained from an industrial brewery. The hopped wort was prepared using 100% Canadian 2-row malt to an original gravity of 12.5º Plato (1.050 specific gravity). The wort was sterilized prior to use at 121ºC for 20 minutes, and cooled to the desired fermentation or propagation temperature overnight.
Propagation and Fermentation
Colonies from WLN plates were inoculated into 5 mL of YPD and grown at 30ºC, 170 rpm for 24 hours. The YPD cultures were transferred into 50 mL of sterilized wort and grown at 30ºC, 170 rpm for 24 hours. Lab-scale fermentations were carried out in triplicate at 30°C for 12 days. The wort cultures described above were counted using a haemocytometer and inoculated at a rate of 1.2x107 cells/mL into 70 mL of sterilized wort in ‘spice jars’ fitted with airlocks. The jars were incubated without shaking to best approximate typical beer fermentation conditions. Fermentation profiles were acquired by weighing the spice jars to measure CO2 release, normalizing against water evaporation from the airlocks.
Beer Metabolite Analysis
Following fermentation, samples were collected and filtered using a 0.45 µm syringe filter prior to metabolite analysis. Flavour metabolite analysis was performed using HS-SPME-GC-MS, with a method adapted from Rodriguez-Bencomo et al., 2012. The optimized final conditions were as follows: 2 mL of beer sample, 0.6 g of NaCl, 10 µL of 3-octanol (0.01 mg/mL), and 10 µL of 3,4-dimethylphenol (0.4 mg/mL). 3-octanol and 3,4-dimethylphenol were used as internal standards.
In addition, the ethanol concentration was measured using HPLC and a RI detector. The samples were run on an Aminex HPX-87H column, using 5 mM sulfuric acid as the mobile phase, under the following conditions: flow rate of 0.6 mL/min, 620 psi, and 60°C. The sample contained 400 µL of filtered beer and 50 µL of 6% (v/v) isopropanol as the internal standard.
Multivariate Analysis
RP
Phenotypic Assays
Temp tolerance - Caroline
To determine ethanol tolerance, yeast cultures grown for 24h at 170rpm at 30ºC in YPD were inoculated into YPD with increasing concentrations of ethanol (YPD + EtOH 5%; 7%; 9%; 11%; 12%; 13%; 14%; 15%) to an initial density of 0.1 OD600 in 150µL media in sterile 96 well plates, and grown with shaking at 30ºC for 24h before being assessed visually for growth.
To determine flocculation, yeast cultures were grown for 24h at 170rpm at 30ºC in YPD, and then 0.5mL was inoculated into 5mL sterilized wort, which was then grown for 24h at 170rpm at 30ºC. Flocculation was assessed using the spectrophotometric absorbance methodology of ASBC method Yeast-11. Values are expressed as % flocculance, with <20% representing non-flocculant yeast and >85% representing highly flocculant yeast