Team:Freiburg/Notebook/21 July

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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 21 July </a>
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*third another GST affinity purification -> Precipitator will be in the flowthrough
*third another GST affinity purification -> Precipitator will be in the flowthrough
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{{:Team:Freiburg/Templates/footer}}

Latest revision as of 00:59, 22 September 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!


Precipitator

Precipitator Ruediger


inoculated cells from yesterdays plating. All 12 plates had colonies. I picked 2 from each plate. Incubation at 37°C overnight at 300rpm.


further Project plan

  • if GFP Pdb works, I will pick best affinity Pbd and use it for the precipitator.
  • I will also need a primer for the Precipitator with a GST tag and a TEV cleavage site[http://en.wikipedia.org/wiki/TEV_protease] inbetween.
  • Need to order a GST column (GST-4B) for the ÄKTA as well as PreScission Protease GST
  • check toolbox plasmid for pGEX-6P
  • check affinity calculation programs Scatchand plots and Klötz (to estimate Ni-Precipitator binding)
    • data for these calculations could be generated with ultrafiltration of the protein bound to nickel, to see how much nickel goes through - can be tested chemically
  • 300ml E.coli culture needed for protein purification in ÄKTA
  • first there will be the GST affinity purification of the lysate of the E. coli culture -> Precipitator will be in the eluate
  • second a digestion of the eluate with the Cleavage enzyme
  • third another GST affinity purification -> Precipitator will be in the flowthrough

                       

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