Team:UT-Tokyo/Data/Method

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Reagents

SOB broth

reagents final conc. amount
Bacto trypton 2% 20g
NaCl 0.05% 0.5g
Yeast extracs 0.5% 5g
KCl (250mM) 2.5mM 10ml
MilliQ - 1000ml
Total 1L

Before use, add 10ml Mg sol.

Mg sol.
reagents final conc. amount
MgCl2(H2O)6 1M 20.33g
MgSO4(H2O)7 1M 24.648g
MilliQ - 100ml
Total 100ml


20× M9 medium

reagents final conc. amount
Na2HPO4 - 6.0g
KH2PO4 - 3.0g
NaCL - 0.5g
NH4Cl - 1.0g
MilliQ - 50ml
Total 50ml

After A.C. , add following reagents to 1L M9 medium

reagents final conc. amount
1M MgSO4 - 1.0ml
2M Glucose - 5.6ml
1% Thiamine - 1.0ml
1M CaCl2 - 0.1ml

LB broth

reagents final conc. amount
Bacto trypton 1% 1g
NaCl 0.5% 0.5g
Yeast extracs 0.5% 0.5g
MilliQ - 100ml
Total 100ml


50× TAE

reagents final conc. amount
Tris 2M 242g
CH3COOH 1M 57.1mL
EDTA (0.5M, pH=8.0) 0.05M 100ml
MilliQ - 1000ml
Total 1L



TB

reagents final conc. amount
KOH 500mM sol. 250mM 242g
PIPES 500mM sol. 10mM 2ml
CaCl2 750mM sol. 15mM 2ml
KCl 2.5M sol. 250mM 10ml
MnCl2 550mM sol. 55mM 10ml
MilliQ - 100ml
Total 100ml

Strains

JM109 (Tkara Bio INC.)

  • Genotype:
recA1, endA1, gyrA96, thi-1, hsdR17(rK-mk+), e14–(mcrA–), supE44, relA1, Δ(lac-proAB)/F' [traD36, proAB+, lac Iq, lacZΔM15]

BL21(DE3)

  • Genotype:
F- ompT hsdSB (rB-mB-) gal dcm (DE3)

ccdB survival (invitrogen)

  • Genotype:
F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG fhuA::IS2

cheZ-

DH5α (invitrogen)

  • Genotype:
F- φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk-, mk+) phoA supE44 λ-thi-1 gyrA96 relA1 tonA

Assembly parts

Digest

Ligation

colony PCR

Transformation

Making Competent E. coli cell

Transformation of E. coli

Purification of DNA

Miniprep

Gel extraction

PCR clean-up

Ethanol precipitation

Analysis of DNA

Gel electrophoresis

Sequencing

Dual luciferase assay

Cell diffsion assay

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