Team:CD-SCU-CHINA/Notebook

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* '''Genome extraction'''
 +
** Using gene extraction kit to isolate the whole genome of Alcanivorax borkumensis SK2<br>
 +
[[File:Genome 8.5.jpg]]
 +
* '''Gene isolate using PCR'''
 +
** Normal primer design and '''gradient PCR''' to amplify the gene.<br>
 +
[[File:Gradent2.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<br>
 +
 +
[[File: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!
 +
[[File:Plasmid ex.jpg]]
 +
* '''Double Digestion for chek'''
 +
 +
* '''Temperament chromatography'''
 +
for the function test of the degradation of the two enzyme gene.

Latest revision as of 03:42, 27 September 2012

Untitled Document

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

Genome 8.5.jpg

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

Gradent2.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
  • 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

  • Double Digestion for chek
  • Temperament chromatography

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

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