Team:Wageningen UR/Protocol

From 2012.igem.org

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= Protocol =
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= Methods =
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The use of Virus-Like-Particles as medicine carrier is new for iGEM. This means the whole production, purification and detection of Virus-Like-Particles is also new in iGEM. In this section we will explain how the different methods work and how it all fits together.
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<ul>
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<li>[[Team:Wageningen_UR/MethodsProduction|Production]]</li>
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<li>[[Team:Wageningen_UR/MethodsPurification|Purification]]</li>
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</ul>
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= Protocols =
== Medium & Buffer recipes ==
== Medium & Buffer recipes ==
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<li>[[Team:Wageningen_UR/Protocol/Reassemblybuffer|Reassembly buffer]]</li>
<li>[[Team:Wageningen_UR/Protocol/Reassemblybuffer|Reassembly buffer]]</li>
<li>[[Team:Wageningen_UR/Protocol/Virusbuffer|Virus buffer]]</li>
<li>[[Team:Wageningen_UR/Protocol/Virusbuffer|Virus buffer]]</li>
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<li>[[Team:Wageningen_UR/Protocol/FormationBufferHepB|Formation Buffer HepBcAg]]</li>
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<li>[[Team:Wageningen_UR/Protocol/WashingBuffer|Washing Buffer HepBcAg]]</li>
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<li>[[Team:Wageningen_UR/Protocol/FormationBufferPolero|Formation Buffer Polero]]</li>
<li>[[Team:Wageningen_UR/Protocol/Towbinselectrotransferbuffer1x|Towbin's electrotransfer buffer 1x]]</li>
<li>[[Team:Wageningen_UR/Protocol/Towbinselectrotransferbuffer1x|Towbin's electrotransfer buffer 1x]]</li>
<li>[[Team:Wageningen_UR/Protocol/Towbinselectrotransferbuffer10x|Towbin's electrotransfer buffer 10x]]</li>
<li>[[Team:Wageningen_UR/Protocol/Towbinselectrotransferbuffer10x|Towbin's electrotransfer buffer 10x]]</li>
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<li>[[Team:Wageningen_UR/Protocol/StartupCCMV|Growing culture]]</li>
<li>[[Team:Wageningen_UR/Protocol/StartupCCMV|Growing culture]]</li>
<li>[[Team:Wageningen_UR/Protocol/DialysisCCMV|Dialysis of the VLPs]]</li>
<li>[[Team:Wageningen_UR/Protocol/DialysisCCMV|Dialysis of the VLPs]]</li>
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<li>[[Team:Wageningen_UR/Protocol/RoundupCCMV|Purifing the VLPs]]</li>
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<li>[[Team:Wageningen_UR/Protocol/RoundupCCMV|Purifying the VLPs]]</li>
</ul>
</ul>
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<li>[[Team:Wageningen_UR/Protocol/StartupHepB|Growing culture]]</li>
<li>[[Team:Wageningen_UR/Protocol/StartupHepB|Growing culture]]</li>
<li>[[Team:Wageningen_UR/Protocol/DialysisHepB|Dialysis of the VLPs]]</li>
<li>[[Team:Wageningen_UR/Protocol/DialysisHepB|Dialysis of the VLPs]]</li>
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<li>[[Team:Wageningen_UR/Protocol/RoundupHepB|Purifing the VLPs]]</li>
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<li>[[Team:Wageningen_UR/Protocol/RoundupHepB|Purifying the VLPs]]</li>
</ul>
</ul>
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== Procedure ==
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== Polerovirus Coat Protein VLP formation ==
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<ol>
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<li>Prepare a Erlenmeyer flask with 50mL of LB-medium and keep it at 37°C</li>
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<li>Pick E.coli BL21 from a plate and grow them over night in a 10 mL LB culture containing 50mg/L Kanamycin at 37°C</li>
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<li>Inoculate the large flask with 1 mL of the overnight culture and grow at 37°C for 2.5h until the OD600 reaches approx. 0.6</li>
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<li>Induce with '''1.25 mM IPTG''' (0.25 mL of a 250 mM stock)</li>
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<li>Incubate at 37°C for 4 h</li>
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<li>Centrifuge the cells in 50ml greiner tubes  at 4700 rpm for 18 minutes</li>
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<li>Decant the supernatant and resuspend the pellets in ± 10 mL washing buffer each</li>
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<li>Centrifuge again at 4700 rpm for 18 minutes</li>
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<li>Decant the supernatant and centrifuge for another minute</li>
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<li>Pipette any liquid to clear the tube of supernatant</li>
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<li>(possible to freeze the cells to -20°C at this point)</li>
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</ol>
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== TuYV Coat Protein VLP formation ==
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== PLRV Coat Protein VLP formation ==
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<ul>
<ul>
<li>[[Team:Wageningen_UR/Protocol/RNA|RNA isolation from potato leaf material]]</li>
<li>[[Team:Wageningen_UR/Protocol/RNA|RNA isolation from potato leaf material]]</li>
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<li>[[Team:Wageningen_UR/Protocol/StartupPolero|Growing culture]]</li>
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<li>[[Team:Wageningen_UR/Protocol/DialysisPolero|Dialysis of the VLPs]]</li>
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<li>[[Team:Wageningen_UR/Protocol/RoundupPolero|Purifying the VLPs]]</li>
</ul>
</ul>
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<li>[[Team:Wageningen_UR/Protocol/Frenchpress|French press user manual]]</li>
<li>[[Team:Wageningen_UR/Protocol/Frenchpress|French press user manual]]</li>
<li>[[Team:Wageningen_UR/Protocol/FPLC|FPLC user manual]]</li>
<li>[[Team:Wageningen_UR/Protocol/FPLC|FPLC user manual]]</li>
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<li>[[Team:Wageningen_UR/Protocol/Mutagenesis|Mutagenesis]]</li>
</ul>
</ul>

Latest revision as of 10:44, 26 October 2012

Contents

Methods

The use of Virus-Like-Particles as medicine carrier is new for iGEM. This means the whole production, purification and detection of Virus-Like-Particles is also new in iGEM. In this section we will explain how the different methods work and how it all fits together.

Protocols

Medium & Buffer recipes

CCMV Coat Protein VLP formation

Hepatitis B Coat Protein VLP formation

Polerovirus Coat Protein VLP formation

General Protocol

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