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

From 2013.igem.org

(Difference between revisions)
Line 59: Line 59:
<h1>Expression of proteins in Escherichia coli</h1>
<h1>Expression of proteins in Escherichia coli</h1>
<p>Protocol：The induction expression isolation and purification of protein in E.coli-BL21
<p>Protocol：The induction expression isolation and purification of protein in E.coli-BL21
-
LB medium (1L)                             Binding buffer(1L)                     Elution Buffer(1L)
+
<table width="580" border="2">
-
Tryptone             10g                   Tris               20mM             Add 500mM iminazole to binding  
+
  <tr>
-
Yeast extract         5g Sodium chloride   500mM             buffer
+
    <td>LB medium (1L)</td>
-
Sodium chloride       5g                   Add HCl or NaOH until the pH is 8.0
+
    <td>Volume</td>
 +
    <td>Binding buffer(1L)</td>
 +
    <td>Volume</td>
 +
    <td>Elution Buffer(1L)</td>
 +
    <td></td>
 +
  </tr>
 +
    <tr>
 +
    <td>Tryptone</td>
 +
    <td>10g</td>
 +
    <td>Tris</td>
 +
    <td>20mM</td>
 +
    <td>Add 500mM iminazole to binding buffer</td>
 +
    <td></td>
 +
  </tr>
 +
  <tr>
 +
    <td>Yeast extract</td>
 +
    <td>5g</td>
 +
    <td>Sodium chloride</td>
 +
    <td>500mM</td>
 +
    <td></td>
 +
    <td></td>
 +
  </tr>
 +
  <tr>
 +
    <td>Sodium chloride</td>
 +
    <td>5g</td>
 +
    <td>Add HCl or NaOH until the pH is 8.0</td>
 +
    <td></td>
 +
    <td></td>
 +
    <td></td>
 +
  </tr>
 +
</table>
 +
 
experimental procedure：
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.
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.

Revision as of 14:30, 25 September 2013

Expression of proteins in Escherichia coli

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

LB medium (1L) Volume Binding buffer(1L) Volume 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/min,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

Retrieved from "http://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/Expression_of_proteins_in_Escherichia_coli"