IGEM:Caltech/2007/Protocols/Flow Cytometry

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(New page: 1. Set up 10mL LB cultures with appropriate antibiotic and aTc concentrations. Grow overnight at 37C while shaking. 2. Take out cultures and measure OD at 600nm. If it's below 0.4, place b...)
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1. Set up 10mL LB cultures with appropriate antibiotic and aTc concentrations. Grow overnight at 37C while shaking.
1. Set up 10mL LB cultures with appropriate antibiotic and aTc concentrations. Grow overnight at 37C while shaking.
 +
2. Take out cultures and measure OD at 600nm. If it's below 0.4, place back at 37C for another 3 hours until it reaches OD ~0.5.
2. Take out cultures and measure OD at 600nm. If it's below 0.4, place back at 37C for another 3 hours until it reaches OD ~0.5.
 +
3. Spin down cultures in 15mL falcons at 10min, 3000xg and 22C.
3. Spin down cultures in 15mL falcons at 10min, 3000xg and 22C.
 +
4. Resuspend in 2mL Bio Assay Buffer (BAB).  
4. Resuspend in 2mL Bio Assay Buffer (BAB).  
 +
5. Spin 2 min to pellet.
5. Spin 2 min to pellet.
 +
6. Resuspend in BAB + antibiotic + aTc (at same antibiotic and aTc concentrations as the original overnight cultures).  
6. Resuspend in BAB + antibiotic + aTc (at same antibiotic and aTc concentrations as the original overnight cultures).  
 +
7. Measure OD and make sure it's between 0.4 and 0.6. If it's below 0.4, spin and pellet and try again.
7. Measure OD and make sure it's between 0.4 and 0.6. If it's below 0.4, spin and pellet and try again.
 +
8. Put in 37C shaker for 4 hours.
8. Put in 37C shaker for 4 hours.
 +
9. Measure fluorescence of 1.5mL samples.
9. Measure fluorescence of 1.5mL samples.

Revision as of 02:34, 9 October 2007

1. Set up 10mL LB cultures with appropriate antibiotic and aTc concentrations. Grow overnight at 37C while shaking.

2. Take out cultures and measure OD at 600nm. If it's below 0.4, place back at 37C for another 3 hours until it reaches OD ~0.5.

3. Spin down cultures in 15mL falcons at 10min, 3000xg and 22C.

4. Resuspend in 2mL Bio Assay Buffer (BAB).

5. Spin 2 min to pellet.

6. Resuspend in BAB + antibiotic + aTc (at same antibiotic and aTc concentrations as the original overnight cultures).

7. Measure OD and make sure it's between 0.4 and 0.6. If it's below 0.4, spin and pellet and try again.

8. Put in 37C shaker for 4 hours.

9. Measure fluorescence of 1.5mL samples.

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