Team:Penn/Protocols
From 2012.igem.org
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<h2>Pelleting & Sonication </h2> | <h2>Pelleting & Sonication </h2> | ||
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<li>Collect cultures by centrifugation for 30 minutes @4°C & 5000g </li> | <li>Collect cultures by centrifugation for 30 minutes @4°C & 5000g </li> | ||
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<ul><li>1 cycle consists of 15 of lysis (10W power) followed by 5 seconds of cool down</li> | <ul><li>1 cycle consists of 15 of lysis (10W power) followed by 5 seconds of cool down</li> | ||
<li>Lysis must be done on ice or excessive protein degradation will occur</li></ul> | <li>Lysis must be done on ice or excessive protein degradation will occur</li></ul> | ||
- | </ | + | <li>Spin lysate down @4°C & 20000g for 30 minutes</li> |
+ | <li>Transfer supernatant into 15 mL Falcon tubes</li> | ||
+ | <li>Add Ni-Agarose beads (1mL of beads per 100mL of culture volume)</li> | ||
+ | <ul><li>Wash beads 2-3 times with equal volumes of RSB250-A</li> | ||
+ | <li>Spin down cells @ ~3000 rpm in a microcentrifuge</li></ul> | ||
+ | <li>Rotate overnight in a 4°C cold room</li> | ||
+ | <li>Pour lysate and beads into a chromatography column, allow to empty through gravity flow</li> | ||
+ | <ul><li>Retain flow through (FT) on ice for later analysis</li></ul> | ||
+ | <li>Wash with 10 mL RSB250-A two times</li> | ||
+ | <ul><li>RSB250-A must be cold<li> | ||
+ | <li>Collect wash fractions for later gel on ice</li></ul> | ||
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Revision as of 05:42, 30 September 2012
Protein Purification
RSB250-A Recipe:
- 20mM Tris
- 250mM NaCl
- 30mM Imidazole
- 10% Glycerol (v/v)
- 20mM Tris
- 250mM NaCl
- 500mM Imidazole
- 10% Glycerol (v/v)
Induction
- Grow a small booster culture in LB Broth overnight (~2-10% of final culture volume) w/ appropriate antibiotics
- Innoculate full scale culture & monitor OD600 every 15-30 minutes until OD600 reaches 0.8
- At OD600=0.8, induce with appropriate concentration of inducer (e.g. 1mM final concentration of IPTG for lac promoter)
- Allow to grow overnight @ 25°C
Pelleting & Sonication
- Collect cultures by centrifugation for 30 minutes @4°C & 5000g
- If sample volume is too large, repeat centrifugation (discarding the supernatant after each centrifugation)
- Resuspend pellet in RSB250-A (minimum 5% of culture volume)
- Pellet must be completely dissolved (no cell clumps) for efficient lysis.
- For especially difficult to resuspend pellets, sucking up and ejecting the cell suspension through a large gauge (narrow) needle several times can break up the pellet effectively
- Sonicate 5 cycles
- 1 cycle consists of 15 of lysis (10W power) followed by 5 seconds of cool down
- Lysis must be done on ice or excessive protein degradation will occur
- Spin lysate down @4°C & 20000g for 30 minutes
- Transfer supernatant into 15 mL Falcon tubes
- Add Ni-Agarose beads (1mL of beads per 100mL of culture volume)
- Wash beads 2-3 times with equal volumes of RSB250-A
- Spin down cells @ ~3000 rpm in a microcentrifuge
- Rotate overnight in a 4°C cold room
- Pour lysate and beads into a chromatography column, allow to empty through gravity flow
- Retain flow through (FT) on ice for later analysis
- Wash with 10 mL RSB250-A two times
- RSB250-A must be cold
- Collect wash fractions for later gel on ice