Drummond:Pregrowth: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
| Line 1: | Line 1: | ||
On Friday: | 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. | *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: | On Monday at 6PM: Let saturate | ||
*Remove plate from incubator. | *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: 2%sucrose, 1%raffinose, 0.5%galactose. THe media for the control experiments is: 2%sucrose, 1%raffinose (no galactose). (Note if you use three strains you will have six falcon tubes) | *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. | *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 of the | *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. | *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: | 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. | *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 500,000 cells. | *Calculate the volume of this saturated culture which contains 500,000 cells. | ||
*Prepare two 50mL tubes for each strain: one with 10mL induction media and one with 10mL control media. | *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 500,000 cells from the corresponding overnight culture. You have already calculated what volume this will be. It may differ for each tube. | ||
*Let grow 8 hours. | *Let grow 8 hours at same temperature as previously with shaking. | ||
* | *This is the final pregrowth step. | ||
On Tuesday at 8PM: | 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. | *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 32,500 cells. | *Calculate the volume of this culture that contains 32,500 cells. | ||
*For a growth experiment, you should have previously made a whole lot of 50mL tubes with 10mL media each*Add 32,500 cells of the reference strain to every 50mL tube. NOTE: If you are also running control experiments, the reference strain should come from media without galactose | *For a growth experiment, you should have previously made a whole lot of 50mL tubes with 10mL media each*Add 32,500 cells of the reference strain to every 50mL tube. NOTE: If you are also running control experiments, the reference strain should come from media without galactose | ||
Revision as of 11:30, 1 May 2009
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 500,000 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 500,000 cells from the corresponding overnight culture. You have already calculated what volume this will be. It may differ for each tube.
- Let grow 8 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 32,500 cells.
- For a growth experiment, you should have previously made a whole lot of 50mL tubes with 10mL media each*Add 32,500 cells of the reference strain to every 50mL tube. NOTE: If you are also running control experiments, the reference strain should come from media without galactose
