Team:CD-SCU-CHINA/Notebook

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Latest revision as of 03:42, 27 September 2012

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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.

@@ Line 1: / Line 1: @@
 {{SCU-IGEM}}
-* Genome extraction
+* '''Genome extraction'''
-* Gene isolate using PCR
+** Using gene extraction kit to isolate the whole genome of Alcanivorax borkumensis SK2<br>
-* Site mutation
+[[File:Genome 8.5.jpg]]
-* Enzyme digest, ligation and transformation
+* '''Gene isolate using PCR'''
-'''* In-fusion cloning and tranformation'''
+** Normal primer design and '''gradient PCR''' to amplify the gene.<br>
-'''* Sequensing'''
+[[File:Gradent2.jpg]]
-'''* Plasmid extraction'''
+* '''Site mutation'''
-'''* Double Digestion for chek'''
+** Using the primer design by us, follow these tips:
-'''* Temperament chromatography
+,Full length not less than 28bp;
-'''
+, 3'end to  mutation site not less than 18bp;
+, 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.