Team:CD-SCU-CHINA/Notebook

From 2012.igem.org

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8/20/12
 +
adh enzyme digestion
 +
20ul 
 +
    1ul XbaI <br>
 +
    1ul PstI <br>
 +
    2ul 10x M buffer <br>
 +
    16ul adh or aldh /20ul  enzyme digestion for 3h<br>
 +
 +
Infusion PCR of oprf
 +
The oprf gene is divided by a EcoRI that is oprf a and oprf b.<br>
 +
Pfu polymerase 0.5ul<br>
 +
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
 +
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>
 +
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
 +
Gnt PCR products gel recovery
 +
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
 +
 +
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
 +
RubB oprf Gnt and Alks gene enzyme digestion
 +
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.
 +
 +
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.
 +
 +
Results: bright strips appear between 55-65 of Gnt
 +
but there are no strips of P450 products.
 +
 +
8/25/12
 +
 +
Transformation of oprf ,alks and Gnt to DH-5a bacteria strain.
 +
25ul competent cell with 5ul DNA
 +
 +
Emzyme digestion of RubB (the restriction enzyme site has been mutated)
 +
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

Revision as of 17:07, 26 September 2012

Untitled Document

8/20/12 adh enzyme digestion 20ul 

    1ul XbaI 

    1ul PstI 

    2ul 10x M buffer 

    16ul adh or aldh /20ul   enzyme digestion for 3h

Infusion PCR of oprf The oprf gene is divided by a EcoRI that is oprf a and oprf b.
Pfu polymerase 0.5ul
10x pfu buffer 5ul
dNTP 1ul
template oprf a 8ul

       oprf b 2ul

forward primer 1ul
reverse primer 1ul
ddH2O 31.5 ul

    total  50ul

PCR amplification of Gnt from the genome Pfu polymerase 0.5ul
10x pfu buffer 5ul
dNTP 1ul
template genome 2.5ul
forward primer 1ul
reverse primer 1ul
ddH2O 39ul

    total  50ul

gradient PCR of alks gradient temperature from 55-60 Pfu polymerase 0.5ul
10x pfu buffer 5ul
dNTP 1ul
template genome 2.5ul
forward primer 1ul
reverse primer 1ul
ddH2O 39ul

    total  50ul

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 Gnt PCR products gel recovery 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

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 RubB oprf Gnt and Alks gene enzyme digestion 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.

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.

Results: bright strips appear between 55-65 of Gnt but there are no strips of P450 products.

8/25/12

Transformation of oprf ,alks and Gnt to DH-5a bacteria strain. 25ul competent cell with 5ul DNA

Emzyme digestion of RubB (the restriction enzyme site has been mutated) 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
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