Team:UT-Tokyo/Data/Method
From 2011.igem.org
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:0.25% agar plate is fragile. You should keep it horizontal. | :0.25% agar plate is fragile. You should keep it horizontal. | ||
- | :Incubation at 37 °C can result in not obvious data due to fast growth | + | :Incubation at 37 °C can result in not obvious data due to fast growth of E.coli. |
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Revision as of 10:24, 1 October 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
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.
Analysis of DNA
Gel electrophoresis
Sequencing
Dual luciferase assay
Cell diffsion assay
Preparation
- 0.25% agar LB plate
Protocol
- Pick single colony using a pipetman with sterile tip.
- Inoculate tip with colony into 0.25% agar plate.
- Incubate at Room Temperature (25°C) for 2~3 days.
Notes
- 0.25% agar plate is fragile. You should keep it horizontal.
- Incubation at 37 °C can result in not obvious data due to fast growth of E.coli.
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)