Haynes Lab:Notebook/ASU iGEM

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(Daily Lab Notebook)
(Daily Lab Notebook)
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==Daily Lab Notebook==
==Daily Lab Notebook==
 +
 +
===6-7-12===
 +
* 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)
 +
 +
===6-8-12===
 +
* 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
 +
 +
===6-12-12===
 +
*DH5a Competent Cell Prep
 +
:*Streak plated cells on LB no amp plate, let grow overnight
 +
 +
===6-13-12===
 +
* 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
 +
 +
===6-14-12===
 +
*Competent cell prep
 +
:*Prepared CaCl<sub>2</sub> buffer solution and CaCl<sub>2</sub> glycerol buffer solution
 +
:*Grew seed colony in 400mL LB no amp
 +
 +
===6-15-12===
 +
*Competent cell prep
 +
:*Centrifuged falcon test tubes containing liquid colonies
 +
:*Resuspended in CaCl<sub>2</sub> buffer solution and incubated for 15 mins
 +
:*Centrifuged and resuspended in CaCl<sub>2</sub> glycerol buffer solution
 +
:*Chilled overnight
 +
 +
===6-16-12===
 +
*Competent cell prep
 +
:*Aliquotted 200uL into test tubes
 +
:*Stored in -80C
 +
 +
===6-17-12===
 +
:*Streak plated prepared competent cells on LB no amp plate
 +
::*Colonies observed
 +
 +
===6-19-12===
 +
*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.
 +
 +
===6-20-12===
 +
* 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
 +
 +
===6-21-12===
 +
*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
 +
 +
===6-22-12===
 +
:*Miniprepped liquid cultures: RBS (well 1:1H BBa_B0030) and TetR GFP (well 2:8A Part:BBa_I13522)
 +
 +
===6-26-12===
 +
* 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
 +
 +
===6-27-12===
 +
* 6-26 transformation results:
 +
:* Controls correct
 +
:* 2x terminator: ~19 colonies
 +
:* RNA pol: 1 colony
 +
* Liquid cultures including controls
 +
 +
===6-28-12===
 +
*Miniprepped double terminator (B0017, 2:6K) and T7 RNA polymerase (I715038, 2:15C) liquid cultures
 +
 +
===7-2-12===
 +
*Cleaned up liquid waste
 +
*Made SOB media
 +
*Finalized oligos for magainin construct
 +
 +
===7-3-12===
 +
*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
 +
|-
 +
|colspan="2" style="background-color: #F2F2F2;"|
 +
|}
 +
__NOTOC__
__NOTOC__

Revision as of 18:39, 12 August 2012

ASU iGEM 2012

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Project Description/Abstract

Team Chimeric Reporter

  • Hyder Hussain: Topo target plasmid design and building
  • Abhinav Markus: (new) Split beta gal assay development for the chimeric reporter (biotinylation proof of concept model)
  • Maddie Sands: (new) Split beta gal assay development for the chimeric reporter (biotinylation proof of concept model)
  • Ryan Muller: Inducible Topo chimeric protein production
  • Ethan Ward: Inducible Topo chimeric protein production


Team Cell Surface Reporter

  • Nisarg Patel: Split beta gal assay development for the magainin reporter
  • Ellen Qin: Magainin fusion protein BioBricking


Support

  • Rohit Rajan: Buffers, media (and quality control), preparation of BioBrick registry submissions
  • Melinda Jenner: Buffers, media (and quality control), preparation of BioBrick registry submissions
  • Amanda Ispas: Buffers, media (and quality control), preparation of BioBrick registry submissions


BioBricks

Magainin Reporter

  • Magainin
  • Magainin + 2xGS Linker + His-Tag
  • Bgal fragment (a-1)
  • Bgal fragment (a-4)
  • Bgal fragment (w)
  • Bgal fragment (1-w)
  • Magainin + 2aa GS Linker + His-Tag + 10aa GS Linker + Bgal fragment (a-1)
  • Magainin + 2aa GS Linker + His-Tag + 10aa GS Linker + Bgal fragment (a-4)
  • Magainin + 2aa GS Linker + His-Tag + 10aa GS Linker + Bgal fragment (w)
  • Magainin + 2aa GS Linker + His-Tag + 10aa GS Linker + Bgal fragment (1-w)
  • His-Tag + 10aa GS Linker + Bgal fragment (a-1)
  • His-Tag + 10aa GS Linker + Bgal fragment (a-4)
  • 13. His-Tag + 10aa GS Linker + Bgal fragment (w)
  • 14. His-Tag + 10aa GS Linker + Bgal fragment (1-w)


Science Resources

General Lab Links

Safety

Daily Lab Notebook

6-7-12

  • Transformation (LSE)
  • Transformed DNA:
  • lacZ (well 4:12G, I732019)
  • p + lacO (well 1:6G, R0011)

6-8-12

  • 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

6-12-12

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

6-13-12

  • Transformation (LSE)
  • Transformed DNA:
  • lacZ (well 4:12G, I732019)
  • p + lacO (well 1:6G, R0011)
  • 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

6-14-12

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

6-15-12

  • 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

6-16-12

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

6-17-12

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

6-19-12

  • Transformation (LSE)
  • Transformed DNA:
  • T7 promoter BBa_I712074
  • Constitutive promoter BBa_J23102
  • Made 50 LB Amp plates.

6-20-12

  • 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)

6-21-12

  • 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

6-22-12

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

6-26-12

  • Transformation:
  • Transformed DNA:
  • double terminator (B0017, 2:6K)
  • T7 RNA polymerase (I715038, 2:15C)
  • puc19, negative control

6-27-12

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

6-28-12

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

7-2-12

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

7-3-12

  • 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


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