"

From 2012.igem.org

Revision as of 17:52, 26 September 2012 (view source) Keger (Talk | contribs) ← Older edit		Revision as of 02:45, 27 September 2012 (view source) Keger (Talk | contribs) (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...") Newer edit →
Line 1:		Line 1:
	{{SCU-IGEM}}		{{SCU-IGEM}}
-	8/20/12	+	* Genome extraction
-	adh enzyme digestion 20ul	+	* Gene isolate using PCR
-	1ul XbaI <br>	+	* Site mutation
-	1ul PstI <br>	+	* Enzyme digest, ligation and transformation
-	2ul 10x M buffer <br>	+	* In-fusion cloning and tranformation
-	16ul adh or aldh /20ul  enzyme digestion for 3h<br>	+	* Sequensing
-		+	* plasmid extraction
-	Infusion PCR of oprf	+	* Double Digestion for chek
-	The oprf gene is divided by a EcoRI that is oprf a and oprf b.<br>	+	* Temperament chromatography
-	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 02:45, 27 September 2012

• 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