Sauer:His6 ClpX purification: Difference between revisions
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(New page: Materials Needed: 1000x AMP: 100mg/mL in 70% Ethanol 2 x 1L TB 1M ZnSO4 1M IPTG His-tagged ClpX strain: SJ01 LB Day One 1.) |
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Materials Needed | ==Materials Needed== | ||
1000x AMP: 100mg/mL in 70% Ethanol | 1000x AMP: 100mg/mL in 70% Ethanol | ||
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LB | LB | ||
==Protocol== | |||
1. | ==Day One== | ||
*1. Innoculate 25mL of LB-AMP with His-tagged ClpX strain. | |||
*2. Warn 2x 1L of TB in incubator or warm room at 37 overnight | |||
==Day Two== | |||
*1. Add 1mL of 1000x AMP to each of the TB Flasks. | |||
*2. Innoculate each flask with 10mL of overnight culture | |||
*3. Grow at 37 C until OD is ~1-1.2 (should take ~>3hrs.) | |||
*4. Drop temperature of incubator to <RT | |||
*5. Add 15uL of ZnSO4 | |||
*6. At OD of 1.7-2.0, induce with 0.25mM IPTG (250uL). Make sure flask is cooled to RT) | |||
*7. After 2-3 hrs. move to bottles and spin down in J6 at 4K/ | |||
*8. Decant Supernatant. Immediately Freeze at -80 | |||
Revision as of 08:41, 26 October 2007
Materials Needed
1000x AMP: 100mg/mL in 70% Ethanol 2 x 1L TB 1M ZnSO4 1M IPTG His-tagged ClpX strain: SJ01 LB
Protocol
Day One
- 1. Innoculate 25mL of LB-AMP with His-tagged ClpX strain.
- 2. Warn 2x 1L of TB in incubator or warm room at 37 overnight
Day Two
- 1. Add 1mL of 1000x AMP to each of the TB Flasks.
- 2. Innoculate each flask with 10mL of overnight culture
- 3. Grow at 37 C until OD is ~1-1.2 (should take ~>3hrs.)
- 4. Drop temperature of incubator to <RT
- 5. Add 15uL of ZnSO4
- 6. At OD of 1.7-2.0, induce with 0.25mM IPTG (250uL). Make sure flask is cooled to RT)
- 7. After 2-3 hrs. move to bottles and spin down in J6 at 4K/
- 8. Decant Supernatant. Immediately Freeze at -80
