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Team:CD-SCU-CHINA/Notebook
From 2012.igem.org
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
