Team:Brown-Stanford/Lab/Notebook/Week11

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Latest revision as of 18:11, 28 September 2011

Brown-Stanford
iGEM
Back to
iGEM HQ

August 22, 2011

PowerCell

  • Retransform LuxBrick into TOP10
  • Digest positive controls:
  • EP, XS, ES, XP; run alongside uncut plasmids (in at 1245pm)
  • positive control bands showed up at significantly higher sizes than expected? otherwise, undig controls showed 3 bands (corresponding to three plasmid conformations), digested ones all showed two; same with lux brick

August 23, 2011

PowerCell

  • the TOPO test with Ana may have worked, many small colonies visible on the 45 μl plates. YAAY!
  • Pick several colonies to separate 3ml LB+amp liquid cultures
  • Performed TOPO ligation and transformation with cscB and gfp PCR products.
  • No visible growth on never UVed ligation
  • New Idea! What could be destroying our ligations/transformations is the UV from the TYPHOON! We forgot about this. It blasts at very high intensity UV for three minutes. Come to think of it, none of our transformations that have gone though the typhoon have ever worked, we think. Use just transluminator. Also, goddamn.
  • two divergent workflows - same as before, cutting out typhoon
  • TOPO, for super concentrations. for great success!

August 24, 2011

PowerCell

  • verification PCR of the ana-TOPO liquid culture
  • success!
  • PCR amplification of pSB1C3
  • fail
  • liquid cultures of gfp and cscB
  • miniprep of lux brick cultures
  • 2 preps of 10 mL tubes
  • result: 590 ng/μl and 600 ng/μl
  • can use this to digest out backbone for other ligation reactions because PCR of backbone to use with TOPO didn’t work
  • Inoculated big 10ml Ana TOPO liq cults.
  • All were innoculated from Ana TOPO LC #1
  • Cyrostocked Ana TOPOs
  • cyrostocked all 4 LC’s
  • 50% glycerol - couldn’t find 40%, was in a hurry.
  • Put in brown cryobox with RegoBricks stuff in -80

August 25, 2011

PowerCell

  • verification PCR of GFP and cscB TOPO clones
  • ran gel, positive control worked, bu not bands in any other lanes
  • we think that since we didn’t shake the liquid culture templates, the cells settled and we didnt get enough cells.
  • miniprep of Ana TOPO liq cultures(4, 10mL each)
  • 91 ng/μl, 165ng/μl, 173 ng/μl, 182 ng/μl

August 26, 2011

PowerCell

  • run gel to verify GFP & cscB TOPO PCR
  • 2nd fail
  • no bands where desired
  • seem to be bands down around where the empty vector would show up but hard to say because all we have is 500bp ladder
  • don’t have money to order ladder, etc waiting on Brown iGEM funding approval
  • lab meeting


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