NYMU Taipei/Lab Notes/2007 8 27

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Contents

plasmid concentration after extraction

part concentration (ug/ul) A260 (OD)
pOmpC 0.06 0.006
EYFP 0.3 0.030
ECFP 0.23 0.023
CinR 0.15 0.015
HSL 0.49 0.049
pCinHSL 0.07 0.007
pCinHSLRBS 0.12 0.012
RBS 0.14 0.014
D-term 0.27 0.027
  • p.s.1: the sample is diluted to 200X, and one OD is 50 ug/mL
  • p.s.2: the calculation of concentration should be OD multiplied only by 10, not by 1,000 (It is a mistake)
  • p.s.3: 50 ug/mL = 0.05 ug/uL, and sample is diluted to 200X. Thus the original concentration should be 0.05 ug/uL * 200 = 10 ug/uL for per OD
  • p.s.4: [http://www.eppendorfna.com/products/quant_biophotometer_de.asp Eppendorf®Biophotometer]

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

Others

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

Who attend?

  • 立翔
  • 德銘
  • 惇茹
  • 香君
  • 宥嫺
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