Team:Penn/Protocols
From 2012.igem.org
(Difference between revisions)
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<li>Pellet must be completely dissolved (no cell clumps) for efficient lysis. | <li>Pellet must be completely dissolved (no cell clumps) for efficient lysis. | ||
<ul><li>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</li></ul></li> | <ul><li>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</li></ul></li> | ||
+ | <li>Sonicate 5 cycles</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> | ||
</p> | </p> | ||
Revision as of 05:26, 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