Team:CD-SCU-CHINA/Notebook

From 2012.igem.org

(Difference between revisions)
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{{SCU-IGEM}}
{{SCU-IGEM}}
* '''Genome extraction'''
* '''Genome extraction'''
 +
** Using gene extraction kit to isolate the whole genome of Alcanivorax borkumensis SK2
* '''Gene isolate using PCR'''
* '''Gene isolate using PCR'''
 +
** Normal primer design and '''gradient PCR''' to amplify the gene.
* '''Site mutation'''
* '''Site mutation'''
-
* '''Enzyme digest, ligation and transformation'''
+
** 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'''
* '''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'''
* '''Sequensing'''
-
* '''Plasmid extraction'''
+
Done by company
 +
 
 +
* '''Colony PCR Plasmid extraction'''
 +
Colony PCR sometimes was not as convenient as we thougt.
* '''Double Digestion for chek'''
* '''Double Digestion for chek'''
 +
* '''Temperament chromatography'''
* '''Temperament chromatography'''
 +
for the function test of the degradation of the two enzyme gene.

Revision as of 03:10, 27 September 2012

Untitled Document

  • 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.

  • Double Digestion for chek
  • Temperament chromatography

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

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