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From 2012.igem.org

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

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

• 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

• Sequensing

Done by company

• Colony PCR Plasmid extraction

Colony PCR sometimes was not as convenient as we thougt. Sometimes plasmid extraction is better!

• Double Digestion for chek

• Temperament chromatography

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