Team:Arizona State/Notebook

From 2012.igem.org

Revision as of 01:44, 1 October 2012

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