Difference between revisions of "Team:Northwestern/OMV purification"

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<h1>Adapted Extracellular Vesicle preparation protocol:</h1>
 
<h1>Adapted Extracellular Vesicle preparation protocol:</h1>
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       <li>Grow desired volume of E. coli liquid culture until mid-log phase (OD<sub>600</sub> = 0.5-0.8)</li>
 
       <li>Grow desired volume of E. coli liquid culture until mid-log phase (OD<sub>600</sub> = 0.5-0.8)</li>
 
       <li>Take note of exact OD<sub>600</sub></li>
 
       <li>Take note of exact OD<sub>600</sub></li>
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       <li>Once all the supernatant has been concentrated, if desired, buffer exchange using the same centrifugal filter unit (e.g. with PBS prior to a SDS-PAGE)</li>
 
       <li>Once all the supernatant has been concentrated, if desired, buffer exchange using the same centrifugal filter unit (e.g. with PBS prior to a SDS-PAGE)</li>
 
       <li>Sample is ready for analysis. </li>
 
       <li>Sample is ready for analysis. </li>
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Latest revision as of 22:01, 16 October 2016

Protocols

Adapted Extracellular Vesicle preparation protocol:

  • Grow desired volume of E. coli liquid culture until mid-log phase (OD600 = 0.5-0.8)
  • Take note of exact OD600
  • Pellet the cells gently (approx. 6,000 g) for 5 minutes
  • Recover as much supernatant as possible without disturbing the cell pellet
  • Leave some supernatant behind if necessary
  • Take note of the volume recovered
  • Filter the supernatant using a 0.22 µm syringe filter unit to remove any remaining cells
  • Change unit if and whenever it clogs
  • Equilibrate a 14 kDa centrifugal filter unit with PBS
  • Concentrate the filtered supernatant with the centrifugal filter unit following manufacturer’s instructions
  • Once all the supernatant has been concentrated, if desired, buffer exchange using the same centrifugal filter unit (e.g. with PBS prior to a SDS-PAGE)
  • Sample is ready for analysis.