Team:UT-Tokyo/Data/Method
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Revision as of 03:55, 23 September 2011
Method
iGEM UT-Tokyo
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