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Revision as of 07:07, 30 August 2007 (view source) Lihsiangyen (Talk | contribs) (→first assembly) ← Older edit		Revision as of 07:08, 30 August 2007 (view source) Lihsiangyen (Talk | contribs) (→Others) Newer edit →
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	==Others==		==Others==
-	*Culture tar-EnvZ, pLacIQ in 5 ml LB liquid medium (Kan:20ug/ml)	+	*liquid culture for tar-EnvZ, pLacIQ in 5 ml LB (Kan:20ug/ml)

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Revision as of 07:08, 30 August 2007

• plasmid concentration after liquid culture extraction

part	concentration (ug/ul)
pOmpC	6
EYFP	30
ECFP	23
CinR	15
HSL	49
pCinHSL	7
pCinHSLRBS	12
RBS	14
D-term	27

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Phase 1 assembly

• pOmpC + (RBS+EYFP)

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• 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)