Difference between revisions of "Team:UCopenhagen/Protocols Lipid Bilayers"

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{{UCopenhagen}}
 
{{UCopenhagen}}
 
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<h1>Protocol: supported lipid bilayer experiments</h1>
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<p>iGEM UCopenhagen 2018</p>
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<h2>Lipid film stocks</h2>
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<p>Lipid mixtures containing sphingomyelin (SM), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and cholesterol (Chl) in a ratio of 44:24:12:20 (SM:DOPC:DOPS:Chl) is prepared for subsequent use to make supported lipid bilayer and liposomes. This composition is inspired from Chatterjee and colleagues [1]. </p>
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<p>The dye Atto655 is included in some of the lipid mixtures to enable detection of the lipid bilayers and liposome by fluorescence microscopy. Biotin was also included in some lipid mixtures to allow for attachment of biotin-containing liposomes to microscopy slides covered with NeutrAvidin.</p>
  
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<h1>Supported Lipid Bilayers</h1>
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  <td><strong>Lipid</strong>
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</p>
 +
  </td>
 +
  <td><strong>MW (g/mol)</strong>
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</p>
 +
  </td>
 +
  <td>DOPC
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</p>
 +
  </td>
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  <td>786.113
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</p>
 +
  </td>
 +
  <td>DOPS
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</p>
 +
  </td>
 +
  <td>810.025
 +
</p>
 +
  </td>
 +
  <td>SM
 +
</p>
 +
  </td>
 +
  <td>760.2
 +
</p>
 +
  </td>
 +
  <td>Chl
 +
</p>
 +
  </td>
 +
  <td>386.7
 +
</p>
 +
  </td>
 +
  <td>Atto655
 +
</p>
 +
  </td>
 +
  <td>1366.0
 +
</p>
 +
  </td>
 +
  <td>Biotin DSPE PEG (2000)
 +
</p>
 +
  </td>
 +
  <td>3016.0
 +
</p>
 +
  </td>
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<p> Lipids solubilized in chloroform and methanol should be handled in a fume hood and pipetted using a Hamilton syringe. </p>
 +
<h3>Materials</h3>
 +
<ul>
 +
 
 +
<li>25 mg/mL stocks of each lipid* (referred to as "stock 1" in notebook)
 +
<li>Lipid dye Atto655, 0.5 mg/mL (referred to as "stock 1" in notebook)
 +
<li>Biotin, PEG, 0.5 mg/mL (referred to as "stock 1" in notebook)
 +
<li>Chloroform and methanol**
 +
 
 +
<h1>Overview of SLB protocols</h1>
 
<p>An overview of the protocols used in the Supported Lipid Bilayers experiments.</p>
 
<p>An overview of the protocols used in the Supported Lipid Bilayers experiments.</p>
 
<h2><a href="https://static.igem.org/mediawiki/2018/6/6d/T--UCopenhagen--Protocol_Liposome_preparation_step_by_step_NH_lab.pdf">Liposomes preparation step-by-step</a></h2>
 
<h2><a href="https://static.igem.org/mediawiki/2018/6/6d/T--UCopenhagen--Protocol_Liposome_preparation_step_by_step_NH_lab.pdf">Liposomes preparation step-by-step</a></h2>

Revision as of 10:30, 16 October 2018

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Protocol: supported lipid bilayer experiments

iGEM UCopenhagen 2018

Lipid film stocks

Lipid mixtures containing sphingomyelin (SM), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and cholesterol (Chl) in a ratio of 44:24:12:20 (SM:DOPC:DOPS:Chl) is prepared for subsequent use to make supported lipid bilayer and liposomes. This composition is inspired from Chatterjee and colleagues [1].

The dye Atto655 is included in some of the lipid mixtures to enable detection of the lipid bilayers and liposome by fluorescence microscopy. Biotin was also included in some lipid mixtures to allow for attachment of biotin-containing liposomes to microscopy slides covered with NeutrAvidin.

Lipid

MW (g/mol)

DOPC

786.113

DOPS

810.025

SM

760.2

Chl

386.7

Atto655

1366.0

Biotin DSPE PEG (2000)

3016.0

Lipids solubilized in chloroform and methanol should be handled in a fume hood and pipetted using a Hamilton syringe.

Materials

  • 25 mg/mL stocks of each lipid* (referred to as "stock 1" in notebook)
  • Lipid dye Atto655, 0.5 mg/mL (referred to as "stock 1" in notebook)
  • Biotin, PEG, 0.5 mg/mL (referred to as "stock 1" in notebook)
  • Chloroform and methanol**

    Overview of SLB protocols

    An overview of the protocols used in the Supported Lipid Bilayers experiments.

    Liposomes preparation step-by-step

    Protocol from Nikos Hatzakis’ enzyme lab. Explains the process of making liposomes. When making liposomes in our own lab, we used liquid nitrogen instead of acetone and dry ice.

    Liposome preparation

    Excel sheet to calculate amount of lipid added when making liposomes. Goes with the protocol Liposomes preparation step-by-step.

    Preparation of supported lipid bilayers

    When making supported lipid bilayers it is based on this protocol step 8 to 12.

    Supported lipid bilayers experiments

    Internal protocol for the experiments performed in the confocal microscope with supported lipid bilayers. The protocol contains parts from other external protocols.