From 2007.igem.org

• plasmid concentration after extraction

part	concentration (ug/ul)	A260 (OD)
pOmpC	6	0.006
EYFP	30	0.030
ECFP	23	0.023
CinR	15	0.015
HSL	49	0.049
pCinHSL	7	0.007
pCinHSLRBS	12	0.012
RBS	14	0.014
D-term	27	0.027

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Phase 1 assembly

• Objective: pOmpC + (RBS+EYFP)
• Ligation
  • Insert:vector=3:1
  • Vector: 300ng/ul(2ul pOmpC after enzyme digestion)
  • Insert: 100ng/ul(2ul EYFP after enzyme digestion)
  • Ligase: 1ul
  • Final volume: 10ul
  • RT 3h
• Enzyme Digestion (pOmpC)
  • pOmpC: 14ul
  • SpeI: 1ul
  • PstI: 1ul
  • 10x buffer: 2, 2ul
  • 10x BSA: 2ul
  • Total: 20ul
  • 37°C, 2h
  • Heat inactivation: 65°C 20min
• Enzyme Digestion (EYFP)
  • EYFP: 3ul
  • XbaI: 1ul
  • PstI: 1ul
  • 10x buffer: 3, 2ul
  • 10x BSA: 2ul
  • H2O: 11ul
  • Total: 20ul
  • 37°C, 2h
  • Heat inactivation: 65°C 20min

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Others

• liquid culture for tar-EnvZ, pLacIQ in 5 ml LB (Kan:20ug/ml)

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Who attend?

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