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Team:Penn/Protocols
From 2012.igem.org
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| Line 129: | Line 129: | ||
</ol> | </ol> | ||
<h2>Pelleting & Sonication </h2> | <h2>Pelleting & Sonication </h2> | ||
| + | <ol> | ||
<li>Collect cultures by centrifugation for 30 minutes @4°C & 5000g </li> | <li>Collect cultures by centrifugation for 30 minutes @4°C & 5000g </li> | ||
| + | <ul><li> If sample volume is too large, repeat centrifugation (discarding the supernatant after each centrifugation)</li></ul> | ||
| + | <li>Resuspend pellet in RSB250-A (minimum 5% of culture volume)</li> | ||
| + | </ol> | ||
</p> | </p> | ||
Revision as of 05:21, 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)
