Team:USTC CHINA/Notebook/Protocols/Expression of proteins in Bacillus subtilis

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<h1>Gel Extraction</h1>
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<h1>Expression of proteins in Bacillus subtilis</h1>
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<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
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<p>Induction with IPTG
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Protocol
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- grow appropriate B. subtilis strain overnight in fresh 2xYT medium
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1. Excise gel slice containing DNA fragment of interest.
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- inoculate into fresh 2xYT medium to an OD600 of 0.15
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2. Add 3×sample volume of Buffer DE-A.
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- when culture reaches OD600 0.7 – 0.8, split into 2 portions and induce with 1 mM IPTG
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Incubate at 75° C for 15-20 min or until gel melts completely.
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(CB-0487-1GAM) to one portion (t = 0)
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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- collect samples at different time points for analysis (t = 1,…)
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3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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Repeat wash with Buffer W2
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Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
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5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
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Revision as of 13:43, 25 September 2013

Expression of proteins in Bacillus subtilis

Induction with IPTG - grow appropriate B. subtilis strain overnight in fresh 2xYT medium - inoculate into fresh 2xYT medium to an OD600 of 0.15 - when culture reaches OD600 0.7 – 0.8, split into 2 portions and induce with 1 mM IPTG (CB-0487-1GAM) to one portion (t = 0) - collect samples at different time points for analysis (t = 1,…)

notebook

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