Team:CD-SCU-CHINA/Notebook

From 2012.igem.org

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(Replaced content with "{{SCU-IGEM}} * Genome extraction * Gene isolate using PCR * Site mutation * Enzyme digest, ligation and transformation * In-fusion cloning and tranformation * Sequensing * pl...")
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{{SCU-IGEM}}
{{SCU-IGEM}}
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8/20/12
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* Genome extraction
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adh enzyme digestion 20ul 
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* Gene isolate using PCR
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    1ul XbaI <br>
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* Site mutation
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    1ul PstI <br>
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* Enzyme digest, ligation and transformation
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    2ul 10x M buffer <br>
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* In-fusion cloning and tranformation
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    16ul adh or aldh /20ul  enzyme digestion for 3h<br>
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* Sequensing
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* plasmid extraction
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Infusion PCR of oprf
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* Double Digestion for chek
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The oprf gene is divided by a EcoRI that is oprf a and oprf b.<br>
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* Temperament chromatography
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Pfu polymerase 0.5ul<br>
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10x pfu buffer 5ul<br>
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dNTP 1ul<br>
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template oprf a 8ul<br>
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        oprf b 2ul<br>
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forward primer 1ul<br>
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reverse primer 1ul<br>
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ddH2O  31.5 ul<br>
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    total  50ul<br>
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PCR amplification of Gnt from the genome
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Pfu polymerase 0.5ul<br>
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10x pfu buffer 5ul<br>
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dNTP 1ul<br>
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template genome 2.5ul<br>
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forward primer 1ul<br>
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reverse primer 1ul<br>
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ddH2O  39ul<br>
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    total  50ul<br>
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gradient PCR of alks  gradient temperature from 55-60
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Pfu polymerase 0.5ul<br>
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10x pfu buffer 5ul<br>
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dNTP 1ul<br>
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template genome 2.5ul<br>
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forward primer 1ul<br>
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reverse primer 1ul<br>
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ddH2O  39ul<br>
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    total  50ul<br>
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adh and aldh gel recovery
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8/21/12
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PCR result: Gnt no strip
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Oprf and alks appear the strips in the 6th and 7th lane are brighter.
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gradient PCR of Gnt
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the strips are brighter in the 6th ,7th and 8th lanes.
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8/22/12
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Gnt PCR products gel recovery
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Parts of gnt and alks amplifying through PCR
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Gnt a 0-543  543 bp Gnt b 543-557 14bp Gnt c 557-706 149bp
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Alks
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Alks a 0-1283 1283bp  alks b 1283-2619 1336bp
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Parts of gnt and alks recycle
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8/23/12
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Infusion PCR of gnt and alks
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Pfu polymerase 0.5ul                   
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10x pfu buffer 5ul
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dNTP 1ul
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template gnt a 2ul
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        gnt b 8ul
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        gnt c 2ul
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forward primer 1ul
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reverse primer 1ul
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ddH2O  29.5ul
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    total  50ul
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Pfu polymerase 0.5ul                   
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10x pfu buffer 5ul
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dNTP 1ul
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template alks a 2ul
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        alks b 2ul
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forward primer 1ul
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reverse primer 1ul
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ddH2O  37.5ul
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    total  50ul
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alks gnt infusion PCR products gel recovery
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8/24/12
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RubB oprf Gnt and Alks gene enzyme digestion
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30ul system
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XbaI 1.5ul
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PstI 1.5ul
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10x M buffer 3ul
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DNA  24ul
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Total volume 30ul
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RubB and P450 infusion PCR
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The RubB is divided by three restriction enzyme site to four parts .we define them respectively RubB a ,b ,c and d.
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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
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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:
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XbaI 1.5ul
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PstI 1.5ul
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10x M buffer 3ul
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Infusion RubB 24ul
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Total volume 30ul  digestion for 3h
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Ligation of aldh and RubB reaction system
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                            Aldh        RubB
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10x T4 DNA ligase buffer        2.5ul      2.5ul
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DNA segment                  17ul      10ul
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Plasmid                      4.5ul      4.5ul
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T4 DNA ligase                  1ul        1ul
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DdH2O                                  7ul
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                            Total volume 25ul
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Revision as of 02:45, 27 September 2012

Untitled Document

  • Genome extraction
  • Gene isolate using PCR
  • Site mutation
  • Enzyme digest, ligation and transformation
  • In-fusion cloning and tranformation
  • Sequensing
  • plasmid extraction
  • Double Digestion for chek
  • Temperament chromatography