Team:CD-SCU-CHINA/Notebook

From 2012.igem.org

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{{SCU-IGEM}}
{{SCU-IGEM}}
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8/20/12
+
* '''Genome extraction'''
-
adh enzyme digestion 20ul 
+
** Using gene extraction kit to isolate the whole genome of Alcanivorax borkumensis SK2<br>
-
    1ul XbaI <br>
+
[[File:Genome 8.5.jpg]]
-
    1ul PstI <br>
+
* '''Gene isolate using PCR'''
-
    2ul 10x M buffer <br>
+
** Normal primer design and '''gradient PCR''' to amplify the gene.<br>
-
    16ul adh or aldh /20ul  enzyme digestion for 3h<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>
-
Infusion PCR of oprf
+
  [[File:Colony.jpg]]
-
The oprf gene is divided by a EcoRI that is oprf a and oprf b.<br>
+
* '''Sequensing'''
-
Pfu polymerase 0.5ul<br>
+
Done by company
-
10x pfu buffer 5ul<br>
+
-
dNTP 1ul<br>
+
-
template oprf a 8ul<br>
+
-
        oprf b 2ul<br>
+
-
forward primer 1ul<br>
+
-
reverse primer 1ul<br>
+
-
ddH2O 31.5 ul<br>
+
-
    total  50ul<br>
+
-
PCR amplification of Gnt from the genome
+
-
Pfu polymerase 0.5ul<br>
+
-
10x pfu buffer 5ul<br>
+
-
dNTP 1ul<br>
+
-
template genome 2.5ul<br>
+
-
forward primer 1ul<br>
+
-
reverse primer 1ul<br>
+
-
ddH2O  39ul<br>
+
-
    total  50ul<br>
+
-
gradient PCR of alks  gradient temperature from 55-60
+
* '''Colony PCR Plasmid extraction'''
-
Pfu polymerase 0.5ul<br>
+
Colony PCR sometimes was not as convenient as we thougt. Sometimes plasmid extraction is better!
-
10x pfu buffer 5ul<br>
+
[[File:Plasmid ex.jpg]]
-
dNTP 1ul<br>
+
* '''Double Digestion for chek'''
-
template genome 2.5ul<br>
+
-
forward primer 1ul<br>
+
-
reverse primer 1ul<br>
+
-
ddH2O  39ul<br>
+
-
    total  50ul<br>
+
-
adh and aldh gel recovery
+
-
8/21/12
+
-
PCR result: Gnt no strip
+
-
Oprf and alks appear the strips in the 6th and 7th lane are brighter.
+
-
gradient PCR of Gnt
+
-
the strips are brighter in the 6th ,7th and 8th lanes.
+
-
8/22/12
+
* '''Temperament chromatography'''
-
Gnt PCR products gel recovery
+
for the function test of the degradation of the two enzyme gene.
-
Parts of gnt and alks amplifying through PCR
+
-
Gnt a 0-543  543 bp Gnt b 543-557 14bp Gnt c 557-706 149bp
+
-
Alks
+
-
Alks a 0-1283 1283bp  alks b 1283-2619 1336bp
+
-
 
+
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Parts of gnt and alks recycle
+
-
 
+
-
8/23/12
+
-
Infusion PCR of gnt and alks
+
-
 
+
-
Pfu polymerase 0.5ul                   
+
-
10x pfu buffer 5ul
+
-
dNTP 1ul
+
-
template gnt a 2ul
+
-
        gnt b 8ul
+
-
        gnt c 2ul
+
-
forward primer 1ul
+
-
reverse primer 1ul
+
-
ddH2O  29.5ul
+
-
    total  50ul
+
-
 
+
-
Pfu polymerase 0.5ul                   
+
-
10x pfu buffer 5ul
+
-
dNTP 1ul
+
-
template alks a 2ul
+
-
        alks b 2ul
+
-
forward primer 1ul
+
-
reverse primer 1ul
+
-
ddH2O  37.5ul
+
-
    total  50ul
+
-
 
+
-
alks gnt infusion PCR products gel recovery
+
-
 
+
-
8/24/12
+
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RubB oprf Gnt and Alks gene enzyme digestion
+
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30ul system
+
-
XbaI 1.5ul
+
-
PstI 1.5ul
+
-
10x M buffer 3ul
+
-
DNA  24ul
+
-
Total volume 30ul
+
-
RubB and P450 infusion PCR
+
-
The RubB is divided by three restriction enzyme site to four parts .we define them respectively RubB a ,b ,c and d.
+
-
 
+
-
The infusion PCR of RubB ,I add four parts including RubB a 2ul , RubB b 3ul, RubB c 3ul and RubB d 6ul to the reaction system. And the gradient of annealing temperature is from 55-65
+
-
The P450 is divided to three parts. we define them a ,b ,c.
+
-
 
+
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The infusion PCR of P450 ,I add three parts including a ,b, c,each 6ul to the reaction system. and the gradient is same as the Gnt.
+
-
 
+
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Results: bright strips appear between 55-65 of Gnt
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-
but there are no strips of P450 products.
+
-
 
+
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8/25/12
+
-
 
+
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Transformation of oprf ,alks and Gnt to DH-5a bacteria strain.
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25ul competent cell with 5ul DNA
+
-
 
+
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Emzyme digestion of RubB (the restriction enzyme site has been mutated)
+
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20ul reaction system:
+
-
XbaI 1.5ul
+
-
PstI 1.5ul
+
-
10x M buffer 3ul
+
-
Infusion RubB 24ul
+
-
Total volume 30ul  digestion for 3h
+
-
 
+
-
Ligation of aldh and RubB reaction system
+
-
                            Aldh        RubB
+
-
 
+
-
10x T4 DNA ligase buffer        2.5ul      2.5ul
+
-
DNA segment                  17ul      10ul
+
-
Plasmid                      4.5ul      4.5ul
+
-
T4 DNA ligase                  1ul        1ul
+
-
DdH2O                                  7ul
+
-
                            Total volume 25ul
+

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.