iGEM 2011 — Team Tokyo_Metropolitan

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# Team:Tokyo Metropolitan/Notebook

### From 2011.igem.org

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| [![](https://static.igem.org/mediawiki/2011/5/56/Tokyo_Metropolitan_home.png)](https://2011.igem.org/Team%3ATokyo_Metropolitan) | [![](https://static.igem.org/mediawiki/2011/f/ff/Tokyo_Metropolitan_team.png)](https://2011.igem.org/Team%3ATokyo_Metropolitan/Team) | [![](https://static.igem.org/mediawiki/2011/0/06/Tokyo_Metropolitan_profile.png)](https://igem.org/Team.cgi?year=2011&team_name=Tokyo_Metropolitan) | [![](https://static.igem.org/mediawiki/2011/9/99/Tokyo_Metropolitan_project.png)](https://2011.igem.org/Team%3ATokyo_Metropolitan/Project) | [![](https://static.igem.org/mediawiki/2011/5/53/Tokyo_Metropolitan_parts.png)](https://2011.igem.org/Team%3ATokyo_Metropolitan/Parts) | [![](https://static.igem.org/mediawiki/2011/8/8b/Tokyo_Metropolitan_notebook.png)](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook) | [![](https://static.igem.org/mediawiki/2011/a/a4/Tokyo_Metropolitan_safety.png)](https://2011.igem.org/Team%3ATokyo_Metropolitan/Safety) | [![](https://static.igem.org/mediawiki/2011/5/5d/Tokyo_Metropolitan_attributions.png)](https://2011.igem.org/Team%3ATokyo_Metropolitan/Attributions) |

[![Tokyo Metropolitan Labnotes.png](/wiki/images/f/fa/Tokyo_Metropolitan_Labnotes.png)](/File%3ATokyo_Metropolitan_Labnotes.png)

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| Contents   * [1 Lab Notebook](#Lab_Notebook) * [2 Lab Protocols](#Lab_Protocols)   + [2.1 PCR (PrimeSTAR® HS DNA Polymerase)](#PCR_.28PrimeSTAR.C2.AE_HS_DNA_Polymerase.29)   + [2.2 Electrophoresis](#Electrophoresis)   + [2.3 Ethanol precipitation](#Ethanol_precipitation)   + [2.4 Plasmid Extraction](#Plasmid_Extraction)   + [2.5 Digestion](#Digestion)   + [2.6 Ligation](#Ligation)   + [2.7 Transformation](#Transformation)   + [2.8 Colony PCR(Takara EX® Taq)](#Colony_PCR.28Takara_EX.C2.AE_Taq.29)   + [2.9 Making Medium for Culture](#Making_Medium_for_Culture) |

# Lab Notebook

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| |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | **Augast** | | | | | | | | Sun | Mon | Tue | Wed | Thu | Fri | Sat | |  | [1](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A01) | [2](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A02) | [3](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A03) | [4](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A04) | [5](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A05) | [6](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A06) | | [7](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A07) | [8](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A08) | [9](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A09) | [10](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A10) | [11](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A11) | [12](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A12) | [13](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A13) | | [14](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A14) | [15](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A15) | [16](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A16) | [17](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A17) | [18](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A18) | [19](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A19) | [20](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A20) | | [21](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A21) | [22](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A22) | [23](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A23) | [24](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A24) | [25](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A25) | [26](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A26) | [27](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A27) | | [28](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A28) | [29](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A29) | [30](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A30) | [31](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/A31) |  |  |  | | |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | **September** | | | | | | | | Sun | Mon | Tue | Wed | Thu | Fri | Sat | |  |  |  |  | [1](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S01) | [2](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S02) | [3](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S03) | | [4](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S04) | [5](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S05) | [6](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S06) | [7](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S07) | [8](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S08) | [9](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S09) | [10](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S10) | | [11](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S11) | [12](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S12) | [13](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S13) | [14](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S14) | [15](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S15) | [16](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S16) | [17](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S17) | | [18](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S18) | [19](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S19) | [20](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S20) | [21](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S21) | [22](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S22) | [23](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S23) | [24](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S24) | | [25](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S25) | [26](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S26) | [27](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S27) | [28](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S28) | [29](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S29) | [30](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/S30) |  | | |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | | **Octorber** | | | | | | | | Sun | Mon | Tue | Wed | Thu | Fri | Sat | |  |  |  |  |  |  | [1](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/O1) | | [2](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/O2) | [3](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/O3) | [4](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/O4) | [5](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/O5) | [6](https://2011.igem.org/Team%3ATokyo_Metropolitan/Notebook/O6) |  |  | |

# 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)

1. 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)
2. Dispense PCR solution to PCR tubes.
3. Set PCR tubes on thermal cycler
4. Run PCR

## Electrophoresis

* Gel
* Electrophoresis bath
* Parafilm
* Micro pipette
* TAE Buffer
* 6× Loading Buffer
* DW

1. Set gel in a gel tank. Pour the TAE Buffer into the gel tank.
   * Loading Buffer
   * DNA solution
   * DW
2. Mix these components on a Parafilm by pipetting.
3. Pour the mixture sample into well.
4. Switch on the power-source and run the gel at 100V for 20min.
5. 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

1. Add 0.1 volume 3M Sodium acetate to the nucleic acid sample and vortex.
2. Add 4 µl of Dr. GenTLETM Precipitation Carrier and vortex.
3. Add 2.5 volumes of ethanol and vortex.
4. Centrifuge at 12,000 rpm at 4˚C for 15 min.
5. Discard the supernatant.
6. Rinse the pellet with 70% ethanol and centrifuge again at 12,000 rpm at 4˚C for 5 min.
7. Discard the supernatant and dry.
8. 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)

1. Centrifuge the E-coli pre-culture on 12,000 rpm for 5min, and then discard the supernatant.
2. Add 100µl of the solution (50ｍM glucose; 10mM EDTA; 25ml Tris-HCl(pH 8.0)), and mix it gently
3. Add 200µl of the 0.2N NaOH; 1％ SDS, and mix it. Incubate on ice for 5min.
4. Add 150µl of 3M Potassium Acetate (pH 4.8, 5M Acetic acid; 3M Potassium), and mix it. Incubate #on ice for 5min.
5. Centrifuge on 12,000 rpm for 5min.
6. Take supernatant into Microtube.

## Digestion

* Micro Tube
* Micro pipette
* Heat Block　or Water bus
* Digest Enzyme
* 10×Buffer
* DW

1. Add below components into a tube and mix them.
   * DNA solution
   * DW
   * 10× Buffer
   * Digest enzyme
2. Incubate at 37°C, from 2h to 16h

## Ligation

* Micro Tube
* Micro pipette
* Heat Block
* T4 Ligase
* 10× Buffer
* DW

1. Add below components into a tube and mix them.
   * DNA solution 1
   * DNA solution 2
   * DW
   * 10× Buffer
   * T4 Ligase
2. Incubate at 16°C, from 30min to 1h

## Transformation

* Micro pipette
* Heat Block　or Water bus
* ECOS™ Competent E. coli
* Plate (Antibiotic)
* Spreader

1. Keep competent cells on ice to thaw it.
2. Add plasmid DNA into E.coli cells.
3. Incubate on ice for 5 min.
4. Put tubes into water bath at 42℃ for 45 seconds.
5. Put tubes back on ice for 2 minutes.
6. Add four times volume of LB (with no antibiotic added). Incubate tubes for 30 min at 37℃.
7. Spread about 100 ul of the resulting culture on LB plates
8. 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

1. Add the all following components on ice.
   * DW
   * 10× PCR Buffer
   * dNTP　(2.5mM)
   * Primer(Forward & Reverse)　 (20µM)
   * Template DNA
   * Taq Polymerase
2. Dispense PCR solution to PCR tubes.
3. Pick up a colony and put it into PCR tube.then inoculate to master plate.
4. Set PCR tubes on thermal cycler
5. 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

1. Measure component of medium and mix them in Erlenmeyer flask.
2. Autoclave(121℃,20min).
3. (Add appropriate antibiotics)
4. Dispense medium to test tube or plate.
5. Store at 4℃

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