Difference between revisions of "Team:Evry/Protocols"

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     <div class="page-header">
 
     <div class="page-header">
 
       <h1>Protocols</h1>
 
       <h1>Protocols</h1>
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<p class="text-justify">
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<strong>Western blot protocol</strong>
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</p>
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<p class="text-justify">
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<ul>
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<li>- Grow cells to mid-log phase (OD600 = 0.5-0.6), spin 5 ml culture, obtaon pellet.</li>
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<li>- Resuspend pellet in 250 ul lysis mix</li>
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<li>- BCA pf protein</li>
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<li>- Add loading dye and boil samples 5 minutes</li>
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<li>- Load gel, run at 50V till through stacking gel, then turn up to 100V</li>
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<li>- Put gel in transfer buffer to equilibrate 5 minutes</li>
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<li>- Transfer at 400mA for 60 minutes</li>
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<li>- Wash blot 5 minutes in TBS to remove MeOH</li>
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<li>- Block membrane 1 hour at room temperature or O.N. 4°C</li>
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<li>- Incubate with primary antibody diluted in blockin buffer for 1 hour shaking at room temperature</li>
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<li>- Wash blot 4 times with TBST 5 minutes</li>
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<li>- Incubate with secondary antibody diluted in blockin buffer for 1 hour shaking at room temperature</li>
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<li>- Wash blot 4 times with TBST 5 minutes</li>
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<li>- Mix Supersignal West Dura solutions 1:1 and put on membrane, incubate at room temperature for 5 minutes</li>
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<li>- Remove excess solution, seal and take pictures</li>
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</ul>
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</p>
  
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<p class="text-justify">
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<strong>Bacterial strains</strong>
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</p>
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<p class="text-justify">
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<i>E. coli</i> DH5alpha was used for cloning purpose in all experiments. Two shuttle plasmids for yeast expressing were transformed, pYGG1 containing URA selection and Amp resistance, pYGG1 containing TRP selection marker and Amp resistance. Those plasmids are high copy episomal plasmids, they contain a 2micron origin and GAL1 galactose inducible promoter. Inserts were cloned by Golden Gate assembly.
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</p>
  
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<p class="text-justify">
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<strong>Yeast culture</strong>
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</p>
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<p class="text-justify">
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Strains used was yeast W303 auxotroph for URA, TRP, HIS, LEU and ADE. A complemented media without amino-acids was used to select and maintain the recombinant yeasts. Yeast was grown to exponential mid-log phase for 12 hours, then resuspended in galactose media for 30h of induction at 25°C. In some experiments, yeast was pre-fixed in 0.5 % paraformaldehyde for 30 minutes followed by washing in PBS before DC loading.
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</p>
  
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<p class="text-justify">
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<strong>Cell culture</strong>
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</p>
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<p class="text-justify">
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The T cell hybridoma cell line, B3Z, specific for the OVA 257-264 peptide (SIINFEKL) in the context of Kb, was a gift from the Curie Institute (Paris V). B3Z were maintained in a medium (RP-10) consisting of RPMI 1640 supplemented with 10% FCS, Glutamax, HEPES,  50 μM 2-mercaptoethanol, 50 U/ml penicillin, and 50 μg/ml streptomycin. All cells were incubated at 37°C with 5% CO2.
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</p>
  
  

Revision as of 00:50, 19 September 2015

   

Protocols

Western blot protocol

  • - Grow cells to mid-log phase (OD600 = 0.5-0.6), spin 5 ml culture, obtaon pellet.
  • - Resuspend pellet in 250 ul lysis mix
  • - BCA pf protein
  • - Add loading dye and boil samples 5 minutes
  • - Load gel, run at 50V till through stacking gel, then turn up to 100V
  • - Put gel in transfer buffer to equilibrate 5 minutes
  • - Transfer at 400mA for 60 minutes
  • - Wash blot 5 minutes in TBS to remove MeOH
  • - Block membrane 1 hour at room temperature or O.N. 4°C
  • - Incubate with primary antibody diluted in blockin buffer for 1 hour shaking at room temperature
  • - Wash blot 4 times with TBST 5 minutes
  • - Incubate with secondary antibody diluted in blockin buffer for 1 hour shaking at room temperature
  • - Wash blot 4 times with TBST 5 minutes
  • - Mix Supersignal West Dura solutions 1:1 and put on membrane, incubate at room temperature for 5 minutes
  • - Remove excess solution, seal and take pictures

Bacterial strains

E. coli DH5alpha was used for cloning purpose in all experiments. Two shuttle plasmids for yeast expressing were transformed, pYGG1 containing URA selection and Amp resistance, pYGG1 containing TRP selection marker and Amp resistance. Those plasmids are high copy episomal plasmids, they contain a 2micron origin and GAL1 galactose inducible promoter. Inserts were cloned by Golden Gate assembly.

Yeast culture

Strains used was yeast W303 auxotroph for URA, TRP, HIS, LEU and ADE. A complemented media without amino-acids was used to select and maintain the recombinant yeasts. Yeast was grown to exponential mid-log phase for 12 hours, then resuspended in galactose media for 30h of induction at 25°C. In some experiments, yeast was pre-fixed in 0.5 % paraformaldehyde for 30 minutes followed by washing in PBS before DC loading.

Cell culture

The T cell hybridoma cell line, B3Z, specific for the OVA 257-264 peptide (SIINFEKL) in the context of Kb, was a gift from the Curie Institute (Paris V). B3Z were maintained in a medium (RP-10) consisting of RPMI 1640 supplemented with 10% FCS, Glutamax, HEPES, 50 μM 2-mercaptoethanol, 50 U/ml penicillin, and 50 μg/ml streptomycin. All cells were incubated at 37°C with 5% CO2.

The protocols we used during the competition are regrouped and documented here.

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