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Team:UT-Tokyo/Data/Method
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Revision as of 03:46, 29 September 2011
Method
iGEM UT-Tokyo
Method
Assembly parts
Digest
Ligation
colony PCR
Transformation
Making Competent E. coli cell
Transformation of E. coli
Preparation
- iGEM parts / ligation products
- SOC or LB (No antibiotic) 500μL
- TE 15μL
- plates
- ice box
- heat block(42°C)
- competent cells
→ always on ice! Melt on ice! Mix DNA as soon as cells melt!
Protocol
to thaw out igem parts
- With a pipette tip, punch a hole in the foil
- Add 15uL of TE (MilliQ),and pipetting
- Pipette 1uL of the resuspended DNA Transformation into your desired competent cells
- Hold on ice for 30 mins
- Heat shock at 42°C for 45 seconds (and on ice after it)
- Add 300uL of LBborth in each epp
- Wait for 10 mins
- Hold at 37℃ for 30 mins
- (this step can be skipped with ampicillin selection)
- Plate out
- Incubate at 37°C
Purification of DNA
Miniprep
Preparation
- kit of Promega (SVMinipreps)
- incubative tube
- 1.5ml epp tube
- MilliQ
Procedure
- pour contents out of the incubative tube into the 1.5ml tube as you can
- centrifuge for 10min (15,000rpm)
- (you can centrifuge incubative tube directly when it can endure up to 6,000g )
- throw supernatant fluid away not to damage the precipitation
- ( you should decant by using yellow tip first / remove culture medium as you can / throw waste water away in bio hazard!)
- add 250μl cell resuspension solution (red label)、suspend completely
- (incomplete suspending decreases yields / you should use epp stand like a washboard)
- add 250μl Cell lysis solution（green label）
- turn the tube upside down four times slowly not to bubble
- add 10μl Alkalin Protease Sol. （small bottle）
- turn the tube upside down four times slowly not to bubble
- wait for 5min （Be careful not to exceed 5min! colon bacillus will disintegrate too much!）
- add 350μl Neutralization Sol. (blue label）
- turn the tube upside down four times slowly not to bubble
- centrifuge for 10min (15,000rpm)
- put the supernatant fluid to column (germ’s wreckage is adhering below)
- centrifuge for 1min (15,000rpm)
- throw flow through (the liquid in the tube below) away
- add 750μl Wash Sol. to column and centrifuge for 1min (15,000rpm)
- throw flow through away, put 250μl Wash Sol. to column and centrifuge for 1min (15,000rpm)
- change the column into 1.5ml tube and centrifuge for 2min (15,000rpm)
- change the tube into new one and add 50μl MilliQ
- (use Nucleas-Free Water in the kit instead of MilliQ)
- centrifuge for 1min (15,000rpm) after waiting for 1min
- take 1 to 1.5μl and determine the concentration by NanoDrop (Don’t dilute)
- label them
Gel extraction
PCR clean-up
Ethanol precipitation
Analysis of DNA
Gel electrophoresis
Sequencing
Dual luciferase assay
Cell diffsion assay
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 (Takara 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
JW 1970 (cheZ-)
derived from K12 BW25113
- Genotype
- (araD-araB)567, lacZ4787(::rrnB-3), lambda-, rph-1, (rhaD-rhaB)568, hsdR514
- details available at http://ecoli.aist-nara.ac.jp/GB6/info.jsp?id=JW1870
DH5α (invitrogen)
- Genotype:
- F- φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk-, mk+) phoA supE44 λ-thi-1 gyrA96 relA1 tonA
