Team:UT-Tokyo/Data/Method
From 2011.igem.org
Method
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iGEM UT-Tokyo
Dual luciferase assay
Preparation
- Dual-GloR Luciferase Assay System (Promega)
- Transfer the contents of one bottle of Dual-GloR Luciferase Buffer to one bottle of Dual-GloR Luciferase Substrate to make Dual-GloR Luciferase Reagent.
- Calculate the amount of Dual-GloR Stop & GloR Reagent needed for the experiment. Using a new container, dilute the Dual-GloR Stop & GloR Substrate 1 : 100 into Dual-GloR Stop & GloR Buffer to make the needed volume of Dual-GloR Stop & GloR Reagent.
- PBS
- Luminescence vial
Protocol
- Centrifuge the incubative tube at 3,000g for 15 min with soft brake.
- Decant supernatant, wash with 1mL PBS
- Centrifuge at 3,000g for 5 min with soft brake.
- Decant supernatant, add 100 ul cell lysis buffer.
- Remove lysate 10-50 ul to the vial.
- Add a volume of Dual-Glo® Reagent equal to the volume of cell lysate.
- Wait at least 5 minutes to allow for chemiluminescence to become stable, then measure the firefly luminescence in a uminometer.
- Add a volume of Dual-Glo® Stop & Glo® Reagent equal to the original culture medium volume.
- Wait at least 5 minutes, then measure Renilla luminescence
- Calculate the ratio of luminescence from the experimental reporter to luminescence from the control reporter.
Notes
Cell diffsion assay
Preparation
- 0.25% agar LB plate (0, 1, 10, 40 or 100uM IPTG)
Protocol
- Pick single colony(cheZ-, cheZres or cheZrep) using a pipetman with sterile tip.
- cheZres is the cheZ- transformed with lacP-RBS-CheZ-d.Ter.
- cheZrep is the cheZ- transformed with cIP-RBS-CheZ-d.Ter-lacP-RBS-cI Represser-d.Ter.
- Inoculate tip with colony into 0.25% agar plates (0, 1, 10, 40 or 100uM IPTG).
- Incubate at room temperature (25°C).
- Exposure the plates every 24 hours.
- Pick single colony(cheZ-, cheZres or cheZrep) using a pipetman with sterile tip.
Notes
- 0.25% agar plate is fragile. You should keep it horizontal.
L-aspartate chemotaxis assay
Preparation
- 0.25% agar LB plate
- 10mM L-Asp solution
Protocol
- Pick JM109 single colony using a pipetman with sterile tip.
- Inoculate tip with the colony into the point that is 25mm distant from the center of 0.25% agar LB plate.
- Incubate at room temperature (25°C) for 45 hours.
- Trickle 40uL of 10mM L-Asp solution in its center(Asp+) or 40uL of MilliQ(Asp-).
- Exposure the plates after inoculating at room temperature for more 20 hours.
Notes
- 0.25% agar plate is fragile. You should keep it horizontal.
SOS response preparation
Preparation
- an UV irradiator (15W / wavelength: 254nm)
- LB broth
- 35mm dish
Protocol
- Culture until OD600 is 0.4-0.6
- Spit into dishes so that a medium depth is 4mm (about 1.4mL in a 35mm dish)
- UV irradiation (15W / 254nm) for 30-60 sec agitating gently.
- Transfer into tubes (1.25mL/tube) and add 2.5mL LB broth.
- Culture for 30mm at 37 °C.
Notes
- Make sure that cells do not lack RecA.
Assembly parts
Digest
Preparation
- Plasmid
- 10x buffer (H or M)
- 0.1% BSA
- Emzyme (EcoR I, Xba I, Spe I, Pst I)
Protocol
- Add plasmid (≦ 2000ng total)
- MilliQ up to 30uL
- 3uL 10x H or M buffer
- 3uL 0.1% BSA
- 0.5uL emzyme I
- 0.5uL emzyme II
- Incubation at 37 °C for more than one hour.
- Add plasmid (≦ 2000ng total)
Notes
- H buffer for E, P, ES, SP and EP cut.
- M buffer for X, S, EX and XP cut.
Ligation
Preparation
- Vector DNA
- Insert DNA
- 2x Ligation Mix (from Ligation Convenience Kit by Nippon Gene)
Protocol
- Make reaction liquid
- MilliQ up to 20uL
- 10uL 2x Ligation Mix
- Vector DNA
- Insert DNA
- Incubation at 16 °C for 15-30 min.
- Make reaction liquid
Notes
- Vector DNA : Insert DNA (molar ratio) = 1 : 1-1.5
- DNA is about 25ng total
Transformation
Making Competent E. coli cell
Preparation
- LB broth
- SOB broth
- Mg sol.
- TB
- DMSO
Protocol
- Shaking culture in 2mL LB broth from frozen stock (37 °C O.N.)
- Add 1.0mL O.N. cuture to 80mL SOB broth(+0.8mL Mg sol.)
- Culture until OD600 is 0.3-0.5 (37 ° for about 2 hours or 20 ° for about 6 hours).
- On ice for 10 min.
- Split into two of 50mL tubes and centrifuge for 5 min (4 °C 4000G).
- Remove supernatant.
- Suspend in TB(10 mL / tube).
- On ice for 15 min.
- Add 200mL DMSO /tube and mix on ice.
- On ice for10-15 min.
- Bring together in a tube and mix well.
- Split into tubes (100uL/tube) and quick freezing in liquid nitrogen.
- strage at -80 °C
Notes
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 15μL of TE (MilliQ),and pipetting
- Pipette 1μL of the resuspended DNA Transformation into your desired competent cells
- Hold on ice for 30 min.
- 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°C for 30 min.
- (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
Preparation
- Kit of promega
- Gel
Procedure
- Gel Slice and PCR Product Preparation
- Dissolving the Gel Slice
- Cut out gel with wanted band and put it in a tube.
- Add 3 parts Mem. binding sol. to 1 part Gel volume.
- Processing PCR Amplifications
- Add an equal volume of Membrane Binding Solution to the PCR amplification.
- Shake & Incubate at 50-65 degrees C until gel is completly dissolved.
- Put in the column.
- Centrifuge at 15,000 rpm for 1 minute
- Empty collection tube and add 700 ul Mem. Wash sol, centrifuge for 1 minute.
- Empty collection tube and add 500 ul Mem. Wash sol, centrifuge for 1 minute.
- Change the column to a new 1.5 ml Eppendorf and centrifuge at 15,000 rpm for 1 minute.
- Change the old tube to a new 1.5 ml Eppendorf, add 30ul Nuclease-Free Water, incubate at RT for 1 minutes, then centrifuge at 15,000 rpm for 1 minute.
- Measure concentration, label the Eppendorf.
- Gel Slice and PCR Product Preparation
Notes
- Percent Recovery Versus Double-Stranded DNA Fragment Size
DNA Fragment Size Percent Recovery 55bp 26% 100bp 84% 1,000bp 92% 23,130bp 47%
Ethanol precipitation for Parts shipping
Preparation
- 100%, 70% Ethanol
- TE buffer
- Miniprep products
Procedure
- Add 2 volumes ice cold absolute ethanol to sample.
- Incubate 1 hr at -80°C.
- (The long incubation time is critical for small fragments)
- Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).
- Remove supernatant.
- Wash with 750-1000 ul room-temperature 95% ethanol.
- Centrifuge for 10 minutes at 4°C at maximum speed (generally >10000 g at least).
- Dry the pellet. For this you can air dry (tubes open, ~15 min).
- (Overdrying can make DNA hard to re-dissolve)
- Add 10 ul TE buffer. Vortex and spin down to resuspend.
- Determine the concentration by NanoDrop.
Notes
- Pellets can be very small and hard to see. Make sure that you don't break invisible pellets on all steps.
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
CJ236 (Takara Bio INC.)
- Genotype:
- dut1, ung1, thi-1, relA1/pCJ105(F' camr)