iGEM 2011 — Team KAIT_Japan

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# Team:KAIT Japan/Notebook

### From 2011.igem.org

無題 2

![](https://static.igem.org/mediawiki/2011/1/1f/KAIT_Japan_ue.png)

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無題 1

## Protocol

**PCR**

1.Add 100μL of reagent solution(Table) to microtube.
2.Dispense 20μL PCR solution to PCR tube.
3.Amplifty target DNA with PCR program.
4.Confirm the band of DNA by agar gel electrophoresis.

|  |  |
| --- | --- |
| reagent name | Volume(μL) |
| TaKaRa Ex Taq | 0.5 |
| 10×Ex Taw buffer | 10 |
| dNDP Mixture | 8 |
| template DNA | 2 |
| fowerd primer | 4 |
| reverse primer | 4 |
| sterile water | 71.5 |
| total | 100 |

**Transformation**
**Preparation of E. coli contain particular plasmid**

1.Incubate frozen competent cell on the ice.
2.Add 2μL of plasmid to competent cell.
3.Incubate for 15 minutes on the ice.
4.Incubate for 45 seconds at 42°C.
5.Incubate for 2 minutes on the ice.
6.Add 250μL LB medium and cultivate for 1 hour at 37°C.
7.Spread culture medium on LB agar plate with appropriate antibiotic.
8.Incubate over night at 37°C.

**Restriction enzyme digestion of DNA**

1.Mix DNA and restriction enzyme(Table).
2.Incubate over night at 37°C.
3.Confirm the band of DNA by agar gel electrophoresis.

|  |  |
| --- | --- |
| reagent name | Volume(μL) |
| DNA | 15 |
| EcoR I | 1 |
| Xba I | 1 |
| buffer M | 2 |
| steril water | 1 |
| total | 20 |

**Plasmid extraction**
**Preparation of plasmid extracted from E. coli**

1. Pick up single colony from agar plate and cultivate it in
20 mL LB medium containing
  appropriate antibiotic overnight at 37°C.
2. Move the culture medium to 50 mL falcon.
3. Centrifuge for 5 minutes at 6000rpm and 4 °C and discard solution.
4. Add 2 mL Solution1, the pellet.
5. Add 4 mL Solution 2, invert tube and stored 3 minutes on ice.
6. Add 3 mL Solution 3, invert tube and stored 5 minutes on ice.
7. Centrifuge for 10 minutes at 9,500rpm and 4 °C.
10.Take aqueous layer to new 50 mL falcon.
11.Add 40 μL RNase, invert tube and incubate for 30 minutes.
12.Add 2 mL phenol chloroform mixture.
13.Centrifuge for 5 minutes at 9,500rpm and 4 °C.
14.Take supernatant to new 50 mL falcon.
15.Add 3 mL chloroform solution and misce.
16.Centrifuge for 3 minutes at 9,500rpm and 4 °C.
17.Take 3 mL supernatant to new 50 mL falcon.
18.Add 300 μL sodium acetate.
19.Add 7.5 mL 100%ethanol.
20.Centrifuge for 20 minutes at 9,500rpm and 4 °C and discard solution.
21.Add 8 mL 70% ethanol.
22.Centrifuge for 20 minutes at 9,500rpm and 4 °C and discard solution.
23.Add 50 μL TE buffer and tapping.
24.Preserve at freezer.

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