Team:Tokyo Metropolitan/Notebook
From 2011.igem.org
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=Lab Notebook= | =Lab Notebook= | ||
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=Lab Protocols= | =Lab Protocols= | ||
==PCR (PrimeSTAR® HS DNA Polymerase)== | ==PCR (PrimeSTAR® HS DNA Polymerase)== | ||
*Ice | *Ice | ||
- | *Micro pipette | + | *Micro pipette |
+ | *Thermal cycler | ||
*PCR Tube | *PCR Tube | ||
*Micro Tube | *Micro Tube | ||
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- | # | + | #Add following components on ice. |
- | # | + | #*DW |
- | # | + | #*5× PCR Buffer |
- | #* | + | #*dNTP (2.5mM) |
- | #* | + | #*Primer(Forward & Reverse) (20µM) |
- | # | + | #*Template DNA |
- | # | + | #*PrimeSTAR® HS DNA Polymerase (2.5U/μl) |
+ | #Dispense PCR solution to PCR tubes. | ||
+ | #Set PCR tubes on thermal cycler | ||
+ | #Run PCR | ||
+ | |||
==Electrophoresis== | ==Electrophoresis== | ||
*Gel | *Gel | ||
- | * | + | *Electrophoresis bath |
*Parafilm | *Parafilm | ||
*Micro pipette | *Micro pipette | ||
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*6× Loading Buffer | *6× Loading Buffer | ||
*DW | *DW | ||
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- | # | + | #Set gel in a gel tank. Pour the TAE Buffer into the gel tank. |
- | #*Loading | + | #*Loading Buffer |
- | #*DNA | + | #*DNA solution |
- | #* | + | #*DW |
- | # | + | #Mix these components on a Parafilm by pipetting. |
- | # | + | #Pour the mixture sample into well. |
+ | #Switch on the power-source and run the gel at 100V for 20min. | ||
+ | #Observe the band by exposing ultraviolet radiation | ||
==Ethanol precipitation== | ==Ethanol precipitation== | ||
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- | # | + | #Add 0.1 volume 3M Sodium acetate to the nucleic acid sample and vortex. |
- | # | + | #Add 4 µl of Dr. GenTLETM Precipitation Carrier and vortex. |
- | #2. | + | #Add 2.5 volumes of ethanol and vortex. |
- | # | + | #Centrifuge at 12,000 rpm at 4˚C for 15 min. |
- | # | + | #Discard the supernatant. |
- | # | + | #Rinse the pellet with 70% ethanol and centrifuge again at 12,000 rpm at 4˚C for 5 min. |
+ | #Discard the supernatant and dry. | ||
+ | #Dissolve the pellet in sterilized water or TE buffer. | ||
+ | |||
==Plasmid Extraction== | ==Plasmid Extraction== | ||
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*Micro pipette | *Micro pipette | ||
*Centrifuge | *Centrifuge | ||
- | * | + | *50mM glucose ; 10mM EDTA ; 25ml Tris-HCl (pH 8.0) |
*5N NaOH | *5N NaOH | ||
*10% SDS | *10% SDS | ||
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- | # | + | #Centrifuge the E-coli pre-culture on 12,000 rpm for 5min, and then discard the supernatant. |
- | # | + | #Add 100µl of the solution (50mM glucose; 10mM EDTA; 25ml Tris-HCl(pH 8.0)), and mix it gently |
- | #0.2N NaOH; 1% | + | #Add 200µl of the 0.2N NaOH; 1% SDS, and mix it. Incubate on ice for 5min. |
- | #3M Potassium Acetate (pH 4.8) | + | #Add 150µl of 3M Potassium Acetate (pH 4.8, 5M Acetic acid; 3M Potassium), and mix it. Incubate #on ice for 5min. |
- | # | + | #Centrifuge on 12,000 rpm for 5min. |
+ | #Take supernatant into Microtube. | ||
+ | |||
==Digestion== | ==Digestion== | ||
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- | # | + | #Add below components into a tube and mix them. |
- | # | + | #*DNA solution |
+ | #*DW | ||
+ | #*10× Buffer | ||
+ | #*Digest enzyme | ||
+ | #Incubate at 37°C, from 2h to 16h | ||
+ | |||
==Ligation== | ==Ligation== | ||
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+ | #Add below components into a tube and mix them. | ||
+ | #*DNA solution 1 | ||
+ | #*DNA solution 2 | ||
+ | #*DW | ||
+ | #*10× Buffer | ||
+ | #*T4 Ligase | ||
+ | #Incubate at 16°C, from 30min to 1h | ||
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- | |||
==Transformation== | ==Transformation== | ||
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+ | #Keep competent cells on ice to thaw it. | ||
+ | #Add plasmid DNA into E.coli cells. | ||
+ | #Incubate on ice for 5 min. | ||
+ | #Put tubes into water bath at 42℃ for 45 seconds. | ||
+ | #Put tubes back on ice for 2 minutes. | ||
+ | #Add four times volume of LB (with no antibiotic added). Incubate tubes for 30 min at 37℃. | ||
+ | #Spread about 100 ul of the resulting culture on LB plates | ||
+ | #Incubate overnight. | ||
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==Colony PCR(Takara EX® Taq)== | ==Colony PCR(Takara EX® Taq)== | ||
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*Burner | *Burner | ||
*70%EtOH | *70%EtOH | ||
- | *Toothpick | + | *Toothpick |
*PCRTube | *PCRTube | ||
*Micro Tube | *Micro Tube | ||
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+ | #Add the all following components on ice. | ||
+ | #*DW | ||
+ | #*10× PCR Buffer | ||
+ | #*dNTP (2.5mM) | ||
+ | #*Primer(Forward & Reverse) (20µM) | ||
+ | #*Template DNA | ||
+ | #*Taq Polymerase | ||
+ | #Dispense PCR solution to PCR tubes. | ||
+ | #Pick up a colony and put it into PCR tube.then inoculate to master plate. | ||
+ | #Set PCR tubes on thermal cycler | ||
+ | #Run PCR | ||
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==Making Medium for Culture== | ==Making Medium for Culture== | ||
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- | # | + | #Measure component of medium and mix them in Erlenmeyer flask. |
- | # | + | #Autoclave(121℃,20min). |
- | + | #(Add appropriate antibiotics) | |
- | + | #Dispense medium to test tube or plate. | |
- | + | #Store at 4℃ | |
- | # | + | |
- | # | + | |
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Latest revision as of 17:17, 4 October 2011
Contents |
Lab Notebook
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|
|
Lab Protocols
PCR (PrimeSTAR® HS DNA Polymerase)
- Ice
- Micro pipette
- Thermal cycler
- PCR Tube
- Micro Tube
- DW
- 5× PCR Buffer
- dNTP (2.5mM)
- Primer(Forward & Reverse) (20µM)
- Template DNA
- PrimeSTAR® HS DNA Polymerase (2.5U/μl)
- Add following components on ice.
- DW
- 5× PCR Buffer
- dNTP (2.5mM)
- Primer(Forward & Reverse) (20µM)
- Template DNA
- PrimeSTAR® HS DNA Polymerase (2.5U/μl)
- Dispense PCR solution to PCR tubes.
- Set PCR tubes on thermal cycler
- Run PCR
Electrophoresis
- Gel
- Electrophoresis bath
- Parafilm
- Micro pipette
- TAE Buffer
- 6× Loading Buffer
- DW
- Set gel in a gel tank. Pour the TAE Buffer into the gel tank.
- Loading Buffer
- DNA solution
- DW
- Mix these components on a Parafilm by pipetting.
- Pour the mixture sample into well.
- Switch on the power-source and run the gel at 100V for 20min.
- Observe the band by exposing ultraviolet radiation
Ethanol precipitation
- Centrifuge
- Micro Tube
- Micro pipette
- Aspirator
- 99.5%Ethanol
- 3.0M Sodium acetate (pH5.2)
- TE Buffer
- Add 0.1 volume 3M Sodium acetate to the nucleic acid sample and vortex.
- Add 4 µl of Dr. GenTLETM Precipitation Carrier and vortex.
- Add 2.5 volumes of ethanol and vortex.
- Centrifuge at 12,000 rpm at 4˚C for 15 min.
- Discard the supernatant.
- Rinse the pellet with 70% ethanol and centrifuge again at 12,000 rpm at 4˚C for 5 min.
- Discard the supernatant and dry.
- Dissolve the pellet in sterilized water or TE buffer.
Plasmid Extraction
- Ice
- Micro Tube
- Micro pipette
- Centrifuge
- 50mM glucose ; 10mM EDTA ; 25ml Tris-HCl (pH 8.0)
- 5N NaOH
- 10% SDS
- 3M Potassium Acetate (pH 4.8)
- Centrifuge the E-coli pre-culture on 12,000 rpm for 5min, and then discard the supernatant.
- Add 100µl of the solution (50mM glucose; 10mM EDTA; 25ml Tris-HCl(pH 8.0)), and mix it gently
- Add 200µl of the 0.2N NaOH; 1% SDS, and mix it. Incubate on ice for 5min.
- Add 150µl of 3M Potassium Acetate (pH 4.8, 5M Acetic acid; 3M Potassium), and mix it. Incubate #on ice for 5min.
- Centrifuge on 12,000 rpm for 5min.
- Take supernatant into Microtube.
Digestion
- Micro Tube
- Micro pipette
- Heat Block or Water bus
- Digest Enzyme
- 10×Buffer
- DW
- Add below components into a tube and mix them.
- DNA solution
- DW
- 10× Buffer
- Digest enzyme
- Incubate at 37°C, from 2h to 16h
Ligation
- Micro Tube
- Micro pipette
- Heat Block
- T4 Ligase
- 10× Buffer
- DW
- Add below components into a tube and mix them.
- DNA solution 1
- DNA solution 2
- DW
- 10× Buffer
- T4 Ligase
- Incubate at 16°C, from 30min to 1h
Transformation
- Micro pipette
- Heat Block or Water bus
- ECOS™ Competent E. coli
- Plate (Antibiotic)
- Spreader
- Keep competent cells on ice to thaw it.
- Add plasmid DNA into E.coli cells.
- Incubate on ice for 5 min.
- Put tubes into water bath at 42℃ for 45 seconds.
- Put tubes back on ice for 2 minutes.
- Add four times volume of LB (with no antibiotic added). Incubate tubes for 30 min at 37℃.
- Spread about 100 ul of the resulting culture on LB plates
- Incubate overnight.
Colony PCR(Takara EX® Taq)
- Plate
- Ice
- Micro pipette
- Thermal cycler
- Burner
- 70%EtOH
- Toothpick
- PCRTube
- Micro Tube
- Spreader
- DW
- 10× PCR Buffer
- dNTP (2.5mM)
- primer(Forward & Reverse) (20µM)
- Taq Polymerase
- Add the all following components on ice.
- DW
- 10× PCR Buffer
- dNTP (2.5mM)
- Primer(Forward & Reverse) (20µM)
- Template DNA
- Taq Polymerase
- Dispense PCR solution to PCR tubes.
- Pick up a colony and put it into PCR tube.then inoculate to master plate.
- Set PCR tubes on thermal cycler
- Run PCR
Making Medium for Culture
- Erlenmeyer flask
- Graduated cylinder
- Test Tube or Plate
- Autoclave
- Stirrer
- Electronic balance
- Burner or Clean bench
- Yeast Extract
- Peptone
- NaCl
- Antibiotic
- Agar
- Measure component of medium and mix them in Erlenmeyer flask.
- Autoclave(121℃,20min).
- (Add appropriate antibiotics)
- Dispense medium to test tube or plate.
- Store at 4℃