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PROTOCOLS

Below are common techniques we perform in our lab.

Cycloheximide Chase

  1. Measure OD600 of O/N cultures.

  2. Dilute cultures to 0.2 in 10 mL. Allow to grow to mid-log phase (OD600 = 0.8-1.2 or ~6 hours).

  3. Collect 7.5 OD units of each culture in a 15 ml conical tube and centrifuge for 2 minutes at 5000 rpm.

  4. Resuspend each cell pellet in 3 ml pre-warmed 30°C media.

  5. Incubate 5 min at 30°C on a heat block or in water bath. 

  6. Add 50 ÂµL ice-cold 20X Stop mix (200 mM NaN3, 5 mg/ml BSA) to separate microcentrifuge tubes (will use in following steps). Will need 1 tube of stop mix per time point per yeast strain.

  7. After the 5 minute incubation, add cycloheximide to final concentration of 0.25 mg/ml (37.5 Âµl of 20 mg/ml stock in ethanol) to each 3 ml resuspended yeast strain. CHASE HAS STARTED.

  8. Take 950 Âµl (~2.5 OD600) aliquots of cells at each time point and add aliquots to appropriate centrifuge tube containing the 50 Âµl ice-cold 20X Stop mix. Vortex, and place on ice until the end of the chase. Time 0 needs to be collected right after adding the cycloheximide to the cell sample.

  9. Keep samples being chased on the 30°C heating block, and vortex every ~5-10 min. Collect the remaining time points at the correct time.

  10. At the end of chase, centrifuge all cells for 30 sec at 7000 rpm.

  11. Aspirate supernatant and store pellets at -20°C or proceed to cell lysis.

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If you use this protocol in your studies, please cite:

Buchanan BW, Lloyd ME, Engle SM, Rubenstein EM. Cycloheximide chase analysis of protein degradation in Saccharomyces cerevisiae. Journal of Visualized Experiments 110 (e53975), 2016.

Growth Assay

  1. Measure OD600 of O/N cultures.

  2. Determine 0.2 OD units.

  3. In a 96-well plate, add fresh sterile water with calculated 0.2 OD units up to 200 ul for each culture in column 1.

  4. Add 125 ul of fresh sterile water to columns 2-4.

  5. Perform six-fold serial dilutions, transferring 25 ul from the first column to the second, from the second to the third, and the third to the fourth.

  6. Plate 4 ul of each well, in order, onto appropriate plates using template and multichannel pipette (in duplicate).

  7. Incubate plates (when dry) at 30°C.

  8. Image plates each day following growth assay until cell growth is visible up to the last column (keep incubating at 30°C).

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If you use this protocol in your studies, please cite:

Watts SG, Crowder JJ, Coffey SC, Rubenstein EM. Growth-based determination and biochemical confirmation of genetic requirements for protein degradation in Saccharomyces cerevisiae. Journal of Visualized Experiments 96 (e52428), 2015.

Lithium Acetate Transformation of Yeast

  1. Grow yeast cells O/N at 30°C in YPD liquid media.

  2. For each transformation, centrifuge 500 uL of yeast at 5000 rpm for 2 minutes at RT. Remove media supernatant.

  3. Resuspend cell pellet in 1 mL of sterile water, repeat centrifuge, and remove media supernatant.

  4. Resuspend yeast in 50 uL sterile 1X LTE.

  5. To each transformation tube add:

                  4 uL carrier DNA (salmon sperm) (5 mg/ml sperm solution)

                  1 uL DNA from miniprep or 1 uL of sterile water for no DNA control

  1. Mix by vortexing, and incubate at RT for 30 minutes.

  2. Add 400 uL of 40% PEG in 1X LTE, mix by vortexing, incubate at RT for several hours (sometimes O/N).

  3. Spin down cells at 5000 rpm for 2 minutes, remove supernatant.

  4. Resuspend pellet in 50 uL of 1M Sorbitol.

  5. Spread entire suspension onto selective plate, incubate at 30°C for 3-5 days.

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