Team:TorontoMaRSDiscovery/Notebook/October

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Latest revision as of 18:16, 21 October 2009

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Home The Team The Project BioBricks Modeling Bioinformatics Safety Notebook


Contents

October 1, 2009

  1. Miniprepped Tet, 5, EncY, C
  2. Ran minipreps and K plasmid on a gel
    • Besides Tet, all samples looked fine
  3. Poured K plates

October 2, 2009

  1. Digested K, EncY and ran on gel

October 3, 2009

  1. Digested EncY and old Tet plasmid -> ran on gel
    • EncY looked fine, but Tet did not show up on gel
  2. Started overnight culture of Tet

October 4, 2009

  1. Miniprepped, digested Tet and ran on gel
    • Tet was confirmed
  2. gel extracted EncY, Tet and got low yields
  3. Started overnight ligations
    • 1: Digested Tet + EncY + 5
    • 2: Gel extraced Tet + Digested EncY + 5
    • negative controls were Tet plasmid alone

October 8, 2009

October 9, 2009

  1. Miniprepped EncY overnight and digested with E,P
  2. Ran digest on a gel
    • Enc part confirmed
  3. Transfected Enc+5 ligations from Sunday/Monday into competent cells from Invitrogen
    • Used NEB Hight Efficiency Transformation Protocol C2987
      • did one 10-fold dilution
      • transformed 5ul of DNA
      • spread 100ul of cells on plate
      • shaker/incubator speed 232rpm

October 11, 2009

  1. Checked plates, no colonies yet
  2. Picked colonies and started overnights from an old 12E plate that wasn't checked: 12E i,ii,iii

October 13, 2009

  1. 12E i and iii showed growth -> miniprepped
  2. Digested and ran on 1% agarose gel
    • Stained gel for 1 hour in EtBr+buffer
    • A faint ~700 band was observed! This could possibly be the 1+2+Enc insert that we want.
  3. To confirm the insert, we decided to sequence it

October 15, 2009

  1. Primers for sequencing 12E insert was designed and ordered

October 16, 2009

  1. EncY prepped and ready to ship down to MIT to submit part to registry
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