Haynes Lab:Notebook/ASU iGEM

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(Daily Lab Notebook)
Current revision (02:13, 17 August 2012) (view source)
 
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* Melinda Jenner: 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
* Amanda Ispas: Buffers, media (and quality control), preparation of BioBrick registry submissions
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* [https://cfo.asu.edu/ehs Environmental Health & Safety at ASU]
* [https://cfo.asu.edu/ehs Environmental Health & Safety at ASU]
* [http://ehsaweb.asu.edu/ Individual Training Record]: To check your ASU individual safety training record, log in at the EHS Assistant page. Click the "Individual Training Record" link and then enter your ASU ID.
* [http://ehsaweb.asu.edu/ Individual Training Record]: To check your ASU individual safety training record, log in at the EHS Assistant page. Click the "Individual Training Record" link and then enter your ASU ID.
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==Daily Lab Notebook==
 
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===6-7-12===
 
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* Transformation (LSE)
 
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:* Transformed DNA:
 
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::* lacZ (well 4:12G, I732019)
 
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::* p + lacO (well 1:6G, R0011)
 
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:* Cells: neb10beta (donated)
 
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:* Protocol from: http://www.neb.com/nebecomm/products/productc3019.asp
 
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:* Controls: puc19, no DNA (8 plates)
 
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===6-8-12===
 
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* Transformation results:
 
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:* puc19: growth
 
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:* negative control: no growth
 
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:* lacZ, lacO: possible small colonies
 
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* liquid culture in amp media (100 ug / ml):
 
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:* no growth of lacZ, lacO
 
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:* growth of puc19
 
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===6-12-12===
 
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*DH5a Competent Cell Prep
 
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:*Streak plated cells on LB no amp plate, let grow overnight
 
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===6-13-12===
 
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* Transformation (LSE)
 
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:* Transformed DNA:
 
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::* lacZ (well 4:12G, I732019)
 
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::* p + lacO (well 1:6G, R0011)
 
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:* Cells: DH5 alpha (donated)
 
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:* Protocol from: http://openwetware.org/wiki/Haynes:Assembly101 (30 minute transformation)
 
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:* Controls: puc19, no DNA (8 plates)
 
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* Transformation (istb4, Abhi)
 
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:* Transformed DNA:
 
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:* Cells:
 
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:* Protocol from:
 
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:* Controls:
 
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*DH5a Chemically Competent cell prep
 
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:*Grew 2 seed colonies from streak plate in LB no amp
 
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:*Grew controls to test for contamination
 
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::*Both Seed colonies grew, no contamination present
 
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===6-14-12===
 
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*Competent cell prep
 
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:*Prepared CaCl<sub>2</sub> buffer solution and CaCl<sub>2</sub> glycerol buffer solution
 
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:*Grew seed colony in 400mL LB no amp
 
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===6-15-12===
 
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*Competent cell prep
 
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:*Centrifuged falcon test tubes containing liquid colonies
 
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:*Resuspended in CaCl<sub>2</sub> buffer solution and incubated for 15 mins
 
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:*Centrifuged and resuspended in CaCl<sub>2</sub> glycerol buffer solution
 
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:*Chilled overnight
 
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===6-16-12===
 
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*Competent cell prep
 
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:*Aliquotted 200uL into test tubes
 
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:*Stored in -80C
 
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===6-17-12===
 
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:*Streak plated prepared competent cells on LB no amp plate
 
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::*Colonies observed
 
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===6-19-12===
 
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*Transformation (LSE)
 
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:*Transformed DNA:
 
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::*T7 promoter BBa_I712074
 
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::*Constitutive promoter BBa_J23102
 
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:* Cells: DH5 alpha (donated)
 
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:* Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
 
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:* Controls: puc19, no DNA
 
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:* Plated 2 copies of each (100 ul, 250 ul) on LB amp plates.
 
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* Made 50 LB Amp plates.
 
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===6-20-12===
 
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* Plated negative control on LB Amp plate
 
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* Liquid cultures of T7 promoter and constitutive promoter
 
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*Transformation (LSE)
 
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:*Transformed DNA:
 
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::*RBS (well 1:1H BBa_B0030)
 
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::*TetR GFP (well 2:8A Part:BBa_I13522)
 
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:* Cells: DH5 alpha (donated)
 
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:* Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
 
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:* Controls: puc19, no DNA
 
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===6-21-12===
 
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*Made Liquid Cultures of E.coli transformed with RBS B0030
 
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*Made Liquid Cultures of E.coli transformed with TetR GFP
 
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*miniprepped and nanodropped T7 promoter BBa_I712074 and Constitutive promoter BBa_J23102 liquid cultures
 
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* liquid cultures:
 
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:* RBS1
 
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:* RBS2 (duplicate_
 
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:* GFP1
 
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:* puc19
 
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:* negative controls
 
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:* 5 ml LB amp
 
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:* overnight cultures
 
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* replated GFP1 & 2 (duplicates)
 
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*Nanodropped plasmid DNA samples
 
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:*Constitutive promoter 1: __ng/uL
 
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:*Constitutive promoter 2: __ng/uL
 
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:*T7 promoter 1: __ng/uL
 
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:*T7 promoter 2: __ng/uL
 
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===6-22-12===
 
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:*Miniprepped liquid cultures: RBS (well 1:1H BBa_B0030) and TetR GFP (well 2:8A Part:BBa_I13522)
 
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===6-26-12===
 
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* Transformation:
 
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:* Transformed DNA:
 
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::* double terminator (B0017, 2:6K)
 
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::* T7 RNA polymerase (I715038, 2:15C)
 
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::* puc19, negative control
 
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:* Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
 
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:* Cells: dh5a
 
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===6-27-12===
 
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* 6-26 transformation results:
 
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:* Controls correct
 
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:* 2x terminator: ~19 colonies
 
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:* RNA pol: 1 colony
 
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* Liquid cultures including controls
 
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===6-28-12===
 
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*Miniprepped double terminator (B0017, 2:6K) and T7 RNA polymerase (I715038, 2:15C) liquid cultures
 
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===7-2-12===
 
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*Cleaned up liquid waste
 
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*Made SOB media
 
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*Finalized oligos for magainin construct
 
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===7-3-12===
 
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*Autoclaved SOB media
 
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*Added glucose to make SOC media
 
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*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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===7-4-12===
 
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===7-5-12===
 
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===7-6-12===
 
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===7-7-12===
 
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===7-8-12===
 
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===7-9-12===
 
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===7-10-12===
 
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===7-11-12===
 
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===7-12-12===
 
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===7-13-12===
 
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===7-14-12===
 
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===7-15-12===
 
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===7-16-12===
 
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===7-17-12===
 
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===7-18-12===
 
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===7-19-12===
 
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===7-20-12===
 
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__NOTOC__
__NOTOC__

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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)


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