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	{{SCU-IGEM}}		{{SCU-IGEM}}
-	8/20/12	+	* '''Genome extraction'''
-	adh enzyme digestion	+	** Using gene extraction kit to isolate the whole genome of Alcanivorax borkumensis SK2<br>
-	20ul	+	[[File:Genome 8.5.jpg]]
-	1ul XbaI <br>	+	* '''Gene isolate using PCR'''
-	1ul PstI <br>	+	** Normal primer design and '''gradient PCR''' to amplify the gene.<br>
-	2ul 10x M buffer <br>	+	[[File:Gradent2.jpg]]
-	16ul adh or aldh /20ul  enzyme digestion for 3h<br>	+	* '''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	+
-		+
-	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	+

---

Latest revision as of 03:42, 27 September 2012

• 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.