Team:USTC CHINA/Notebook/Protocols/Expression of proteins in Escherichia coli

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<h1>Gel Extraction</h1>
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<h1>Expression of proteins in Escherichia coli</h1>
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<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
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<p><span>Protocol：The induction expression isolation and purification of protein in E.coli-BL21</span></br>
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Protocol
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<table width="580" border="2">
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1. Excise gel slice containing DNA fragment of interest.
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  <tr>
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2. Add 3×sample volume of Buffer DE-A.
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    <td>LB medium (1L)</td>
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Incubate at 75° C for 15-20 min or until gel melts completely.
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    <td>Dosage</td>
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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    <td>Binding buffer(1L)</td>
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<img src="https://static.igem.org/mediawiki/2013/4/4d/2013igemustc-china_Protocol1.png" width="580" height="200" />  
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    <td>Concentration</td>
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3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
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    <td>Elution Buffer(1L)</td>
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    <td></td>
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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  </tr>
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Repeat wash with Buffer W2
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    <tr>
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Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
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    <td>Tryptone</td>
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    <td>10g</td>
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5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
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    <td>Tris</td>
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    <td>20mM</td>
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    <td>Add 500mM iminazole to binding buffer</td>
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    <td></td>
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  </tr>
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  <tr>
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    <td>Yeast extract</td>
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    <td>5g</td>
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    <td>Sodium chloride</td>
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    <td>500mM</td>
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    <td></td>
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    <td></td>
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  </tr>
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  <tr>
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    <td>Sodium chloride</td>
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    <td>5g</td>
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    <td>Add HCl or NaOH until the pH is 8.0</td>
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    <td></td>
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    <td></td>
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    <td></td>
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  </tr>
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</table>
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<span>experimental procedure</span>：</br>
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1.Add 100~200 uL E.coli Bl21 storing in -40℃ 50mL LB medium which has been added 5mg ampicillin and culture the bacterial 12hours in a 37℃ Incubator shaker.</br>
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2.Add all the 50ml LB medium to an 1L LB medium which has been added 100mg ampicillin and culture the bacterial in a 16℃ incubator shaker until the OD is between 1.0 and 1.2.</br>
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3.Add 3300mM IPTG into the medium and culture the bacterial for 20 to 24 hours.</br>
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4.Bacteria Collection：Dispense all the bacterial suspension into several 500mL centrifuge bottles and ensure that the error does not exceed 0.1g.Centrifuge under these conditions:The speed is 6000rpm,the time is 10 minutes and the temperature is 4℃ and discard the supernatant.</br>
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5.Resuspend The bacteria in the centrifuge bottles with 40mL binding buffer and transfer the bacterial suspension to an 80ml beaker for ultrasonic disruption under these conditions:The power is 40%，the total working time is 20min and the on time is 2s while the off time is 4s.</br>
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6.Dispense the bacterial suspension into two 500mL centrifuge bottles and ensure that the error does not exceed 0.1g.Centrifuge under these conditions:The speed is 13000rpm/min,the time is 30 minutes and the temperature is 4℃.</br>
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7.Purify the protein through nickel-affinity chromatography column.</br>
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Latest revision as of 09:10, 27 September 2013

Expression of proteins in Escherichia coli

Protocol：The induction expression isolation and purification of protein in E.coli-BL21

LB medium (1L) Dosage Binding buffer(1L) Concentration Elution Buffer(1L)
Tryptone 10g Tris 20mM Add 500mM iminazole to binding buffer
Yeast extract 5g Sodium chloride 500mM
Sodium chloride 5g Add HCl or NaOH until the pH is 8.0
experimental procedure：
1.Add 100~200 uL E.coli Bl21 storing in -40℃ 50mL LB medium which has been added 5mg ampicillin and culture the bacterial 12hours in a 37℃ Incubator shaker.
2.Add all the 50ml LB medium to an 1L LB medium which has been added 100mg ampicillin and culture the bacterial in a 16℃ incubator shaker until the OD is between 1.0 and 1.2.
3.Add 3300mM IPTG into the medium and culture the bacterial for 20 to 24 hours.
4.Bacteria Collection：Dispense all the bacterial suspension into several 500mL centrifuge bottles and ensure that the error does not exceed 0.1g.Centrifuge under these conditions:The speed is 6000rpm,the time is 10 minutes and the temperature is 4℃ and discard the supernatant.
5.Resuspend The bacteria in the centrifuge bottles with 40mL binding buffer and transfer the bacterial suspension to an 80ml beaker for ultrasonic disruption under these conditions:The power is 40%，the total working time is 20min and the on time is 2s while the off time is 4s.
6.Dispense the bacterial suspension into two 500mL centrifuge bottles and ensure that the error does not exceed 0.1g.Centrifuge under these conditions:The speed is 13000rpm/min,the time is 30 minutes and the temperature is 4℃.
7.Purify the protein through nickel-affinity chromatography column.

Notebook

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