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Team:UT-Tokyo/Data/Method
From 2011.igem.org
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:recA1, endA1, gyrA96, thi-1, hsdR17(rK-mk+), e14–(mcrA–), supE44, relA1, Δ(lac-proAB)/F' [traD36, proAB+, lac Iq, lacZΔM15] | :recA1, endA1, gyrA96, thi-1, hsdR17(rK-mk+), e14–(mcrA–), supE44, relA1, Δ(lac-proAB)/F' [traD36, proAB+, lac Iq, lacZΔM15] | ||
| - | ===BL21=== | + | ===BL21(DE3)=== |
===ccdB survival (invitrogen)=== | ===ccdB survival (invitrogen)=== | ||
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===cheZ-=== | ===cheZ-=== | ||
| + | |||
| + | ===DH5α (invitrogen)=== | ||
==Assembly parts== | ==Assembly parts== | ||
Revision as of 14:13, 22 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)
ccdB survival (invitrogen)
derived from the TOP10 strain, and offer the following features:
- -Resistance to the ccdB gene product
- -Resistance to T1 and T5 phage (tonA)
- -Cleaner preparations of DNA and better results in downstream applications due to the elimination of non-specific digestion by endonuclease I (endA1)
- -Reduced occurrence of non-specific recombination in cloned DNA (recA1)
- 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)
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
