Team:Arizona State/Notebook

From 2012.igem.org

(Difference between revisions)
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==June 28==
==June 28==
* Miniprepped double terminator (B0017, 2:6K) and T7 RNA polymerase (I715038, 2:15C) liquid cultures
* Miniprepped double terminator (B0017, 2:6K) and T7 RNA polymerase (I715038, 2:15C) liquid cultures
 +
 +
==July 2==
 +
* Cleaned up liquid waste
 +
* Made SOB media
 +
* Finalized oligos for magainin construct
 +
 +
==July 3==
 +
* Autoclaved SOB media
 +
* Added glucose to make SOC media
 +
* Nanodropped double terminator (B0017, 2:6K) [DT1: 24.5, DT2: 29.6] and T7 RNA polymerase (I715038, 2:15C) [P1: 64.6, P2: 55.3] liquid cultures
 +
 +
==July 24==
 +
* Plasmids arrived courtesy of University of Pennsylvania School of Medicine
 +
* pET29a vectors containing coding sequence for Topoisomerase mutants CSCS and CSCS D168A described here
 +
 +
==July 25==
 +
* Tranformed CSCS topo 0 plasmid and CSCS D168A topo into DH5a Turbo cells (with neg control and Puc19 neg control)
 +
* Plated on Kanamycin plates

Revision as of 01:44, 1 October 2012


June 2012
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July 2012
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August 2012
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September 2012
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Note from Dr. Haynes: Rohit, please transfer all of the Notebook entries from the OWW Wiki
http://openwetware.org/wiki/Haynes_Lab:Notebook/ASU_iGEM
To this page. Follow the format below. I added a couple of sections to help you get started.

June 07

  • Transformation (LSE)
    • Transformed DNA:
      • lacZ (well 4:12G, I732019)
      • p + lacO (well 1:6G, R0011)
    • Cells: neb10beta (donated)
    • Protocol from: http://www.neb.com/nebecomm/products/productc3019.asp
    • Controls: puc19, no DNA (8 plates)

June 08

  • Transformation results
    • puc19: growth
    • negative control: no growth
    • lacZ, lacO: possible small colonies
  • liquid culture in amp media (100 ug / ml):
    • no growth of lacZ, lacO
    • growth of puc19

June 12

  • DH5a Competent Cell Prep
    • Streak plated cells on LB no amp plate, let grow overnight

June 13

  • Transformation (LSE)
    • Transformed DNA:
      • lacZ (well 4:12G, I732019)
      • p + lacO (well 1:6G, R0011)
    • Cells: DH5 alpha (donated)
    • Protocol from: http://openwetware.org/wiki/Haynes:Assembly101 (30 minute transformation)
    • Controls: puc19, no DNA (8 plates)
  • Transformation (istb4, Abhi)
    • Transformed DNA:
    • Cells:
    • Protocol from:
    • Controls:
  • DH5a Chemically Competent cell prep
    • Grew 2 seed colonies from streak plate in LB no amp
    • Grew controls to test for contamination
      • Both Seed colonies grew, no contamination present

June 14

  • Competent cell prep
    • Prepared CaCl2 buffer solution and CaCl2 glycerol buffer solution
    • Grew seed colony in 400mL LB no amp

June 15

  • Competent cell prep
    • Centrifuged falcon test tubes containing liquid colonies
    • Resuspended in CaCl2 buffer solution and incubated for 15 mins
    • Centrifuged and resuspended in CaCl2 glycerol buffer solution
    • Chilled overnight

June 16

  • Competent cell prep
    • Aliquotted 200uL into test tubes
    • Stored in -80C

June 17

  • Streak plated prepared competent cells on LB no amp plate
    • Colonies observed

June 19

  • Transformation (LSE)
    • Transformed DNA:
      • T7 promoter BBa_I712074
      • Constitutive promoter BBa_J23102
    • Cells: DH5 alpha (donated)
    • Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
    • Controls: puc19, no DNA
    • Plated 2 copies of each (100 ul, 250 ul) on LB amp plates.
  • Made 50 LB Amp plates.

June 20

  • Plated negative control on LB Amp plate
  • Liquid cultures of T7 promoter and constitutive promoter
  • Transformation (LSE)
    • Transformed DNA:
      • RBS (well 1:1H BBa_B0030)
      • TetR GFP (well 2:8A Part:BBa_I13522)
    • Cells: DH5 alpha (donated)
    • Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
    • Controls: puc19, no DNA

June 21

  • Made Liquid Cultures of E.coli transformed with RBS B0030
  • Made Liquid Cultures of E.coli transformed with TetR GFP
  • miniprepped and nanodropped T7 promoter BBa_I712074 and Constitutive promoter BBa_J23102 liquid cultures
  • liquid cultures:
    • RBS1
    • RBS2 (duplicate_
    • GFP1
    • puc19
    • negative controls
    • 5 ml LB amp
    • overnight cultures
  • replated GFP1 & 2 (duplicates)
  • Nanodropped plasmid DNA samples
    • Constitutive promoter 1: __ng/uL
    • Constitutive promoter 2: __ng/uL
    • T7 promoter 1: __ng/uL
    • T7 promoter 2: __ng/uL

June 22

  • Miniprepped liquid cultures: RBS (well 1:1H BBa_B0030) and TetR GFP (well 2:8A Part:BBa_I13522)

June 26

  • Transformation:
    • Transformed DNA:
      • double terminator (B0017, 2:6K)
      • T7 RNA polymerase (I715038, 2:15C)
      • puc19, negative control
    • Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
    • Cells: dh5a

June 27

  • 6-26 transformation results:
    • Controls correct
    • 2x terminator: ~19 colonies
    • RNA pol: 1 colony
  • Liquid cultures including controls

June 28

  • Miniprepped double terminator (B0017, 2:6K) and T7 RNA polymerase (I715038, 2:15C) liquid cultures

July 2

  • Cleaned up liquid waste
  • Made SOB media
  • Finalized oligos for magainin construct

July 3

  • Autoclaved SOB media
  • Added glucose to make SOC media
  • Nanodropped double terminator (B0017, 2:6K) [DT1: 24.5, DT2: 29.6] and T7 RNA polymerase (I715038, 2:15C) [P1: 64.6, P2: 55.3] liquid cultures

July 24

  • Plasmids arrived courtesy of University of Pennsylvania School of Medicine
  • pET29a vectors containing coding sequence for Topoisomerase mutants CSCS and CSCS D168A described here

July 25

  • Tranformed CSCS topo 0 plasmid and CSCS D168A topo into DH5a Turbo cells (with neg control and Puc19 neg control)
  • Plated on Kanamycin plates