iGEM 2012 — Team CD-SCU-CHINA

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# Team:CD-SCU-CHINA/Notebook

### From 2012.igem.org

Untitled Document

* [home](https://2012.igem.org/Team%3ACD-SCU-CHINA)
* [team](https://2012.igem.org/Team%3ACD-SCU-CHINA/Team)
* [project](https://2012.igem.org/Team%3ACD-SCU-CHINA/Project)
* [notebook](https://2012.igem.org/Team%3ACD-SCU-CHINA/Notebook)
* [safety](https://2012.igem.org/Team%3ACD-SCU-CHINA/Safety)
* [parts](https://2012.igem.org/Team%3ACD-SCU-CHINA/Parts)
* [human practice](https://2012.igem.org/Team%3ACD-SCU-CHINA/Human%20practice)
* [attributions](https://2012.igem.org/Team%3ACD-SCU-CHINA/Attributions)

* **Genome extraction**
  + Using gene extraction kit to isolate the whole genome of Alcanivorax borkumensis SK2

[![Genome 8.5.jpg](/wiki/images/8/87/Genome_8.5.jpg)](/File%3AGenome_8.5.jpg)

* **Gene isolate using PCR**
  + Normal primer design and **gradient PCR** to amplify the gene.

[![Gradent2.jpg](/wiki/images/e/e8/Gradent2.jpg)](/File%3AGradent2.jpg)

* **Site mutation**
  + Using the primer design by us, follow these tips:

1,Full length not less than 28bp;
2, 3'end to mutation site not less than 18bp;
3, Mutation site to 5' end not less than 10bp.

* **Gene purification ,Enzyme digest, ligation and transformation**

In our experiment, we found the vector (we used) is hard to digest by two enzyme in the same time, it cost us a long time on the identification the correct plamid, unfortunately, it was frustrating. at last, we follow the advice from the teacher, degest one by one, and have an self-ligation test in order to have the correct digested vector.

* **In-fusion cloning and tranformation**

we use this method for the ligation of 6 genes, but follow the instrction of the Clontech company, we may waste a lot of money on it, so we search methods from the paper, first, we test four-way ligation, however, it turn out to be no colony. so we test three-ligation. it was useful.
Methods are as follows:
Vector: more than 50ng;
Gne inserts: more than 5:1 molar ratio to vector
In-fusion enzyme: 2ul every 10ul reaction system
ddH2O: Add water to 10ul

```
![Colony.jpg](/wiki/images/6/60/Colony.jpg)
```

* **Sequensing**

Done by company

* **Colony PCR Plasmid extraction**

Colony PCR sometimes was not as convenient as we thougt. Sometimes plasmid extraction is better!
[![Plasmid ex.jpg](/wiki/images/c/c3/Plasmid_ex.jpg)](/File%3APlasmid_ex.jpg)

* **Double Digestion for chek**

* **Temperament chromatography**

for the function test of the degradation of the two enzyme gene.

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