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On Friday:Streak Out
- Streak cells to singles on Synthetic Complete Plate (with glucose) from freezer stocks. You will have a minimum of three strains (three plates) corresponding to two test strains and one reference strain. A single experiment requires two strains, a test strain and the reference strain. It is best to do experiments for both test strains (folded and misfolded) at the same time to minimize variables in the growth conditions.
On Monday at 6PM: Let saturate
- Remove plate from incubator.
- Prepare falcon tubes with 2mLs of the media you plan to use for the entirety of the growth rate experiment. You will need two tubes for each strain because you should always run an induced and an uninduced control experiment. The media for the induced experiments is: SC 2%sucrose, 1%raffinose, 0.5%galactose. THe media for the control experiments is: SC 2%sucrose, 1%raffinose (no galactose). (Note if you use three strains you will have six falcon tubes)
- Now you will prepare to inoculate BOTH the induced and uninduced control tubes for each strain with the same single colony. Prepare a 1.5mL tube with 500uL of the control media (SC 2%suc 1%raf NO GAL) for each strain (three strains = three tubes). Label these tubes with the strain name. Inoculate this 500uL with a single colony of the appropriate strain using the SC plates from Friday. Mix well by pipeting or vortexing.
- Using these 500uL cultures, inoculate the induced and uninduced falcon tubes with 100uL each of the appropriate strain. To over clarify, no strains are mixed at this point. Each strain is growing separately to acclimate to the study conditions.
- Place these falcon tubes at 30C (or whichever temperature you intend to use for the entirety of the experiment) with shaking overnight. Allow saturation.
On Tuesday at 10AM: Move to exponential phase
- Using coulter counter, measure cells/mL in your o/n cultures. It will likely be 10^8 if saturated.
- Calculate the volume of this saturated culture which contains 250,000 cells.
- Prepare two 50mL tubes for each strain: one with 10mL induction media and one with 10mL control media. Again, if you have three strains you will have six tubes.
- Inoculate this 10mLs with 250,000 cells from the corresponding overnight culture. You have already calculated what volume this will be. It may differ for each tube.
- Let grow 10 hours at same temperature as previously with shaking.
- This is the final pregrowth step.
On Tuesday at 8PM: Mix cultures
- Measure the cells/mL of your 10mL cultures on the coulter counter. These should be close to 10^6.
- Calculate the volume of this culture that contains enough cells to reach 10^6 by your next measurement point. If this is 12 hours away, calculate 75,000 cells. If this is 14 hours away, calculate 32,500 cells.
- For a growth experiment, you should have previously made a whole lot of 50mL tubes with 10mL media each. Prepareation for a fitness assay is not covered in this protocal.
- Add 75,000 or 32,500 cells (depending on your timing) of the induced reference strain to every induced 50mL tube. Add the same number of cells of the UNinduced reference strain to every UNinduced 50mL tube. (NOTE: This sounds funny because the reference is constituitive and never induced, but the experiments are called the induced cultures versus the UNinduced control cultures, and the reference strain has been pregrown in both induced and UNinduced media.) Now add the same number (75,000 or 32,500) cells of each test strain to the appropriate tubes. (Again, fitness experimental design is not covered in this section, only pregrowth is covered here.)