Engineering BioBrick vectors from BioBrick parts/DNA ligation - Revision history

Reshma P. Shetty at 15:42, 18 March 2008

2008-03-18T15:42:29Z

←Older revision		Revision as of 15:42, 18 March 2008
Line 7:		Line 7:
	*Purified, linearized prefix part (in H<sub>2</sub>O)		*Purified, linearized prefix part (in H<sub>2</sub>O)
	*Purified, linearized suffix part (in H<sub>2</sub>O)		*Purified, linearized suffix part (in H<sub>2</sub>O)
		+	*Sterile 0.6mL plastic tubes

	==Ligation mix==		==Ligation mix==

Reshma P. Shetty at 15:36, 18 March 2008

2008-03-18T15:36:17Z

←Older revision		Revision as of 15:36, 18 March 2008
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	==Procedure==		==Procedure==
		+
	Vortex all reagents before use.		Vortex all reagents before use.

Reshma P. Shetty at 15:36, 18 March 2008

2008-03-18T15:36:07Z

←Older revision		Revision as of 15:36, 18 March 2008
Line 16:		Line 16:

	==Procedure==		==Procedure==
		+	Vortex all reagents before use.

	#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube.		#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube.

Reshma P. Shetty at 15:18, 18 March 2008

2008-03-18T15:18:35Z

←Older revision		Revision as of 15:18, 18 March 2008
Line 8:		Line 8:
	*Purified, linearized suffix part (in H<sub>2</sub>O)		*Purified, linearized suffix part (in H<sub>2</sub>O)

-	==Ligation ~~Mix~~==	+	==Ligation mix==

	*2-4 μL each purified, linearized DNA		*2-4 μL each purified, linearized DNA

Reshma P. Shetty at 15:18, 18 March 2008

2008-03-18T15:18:12Z

←Older revision		Revision as of 15:18, 18 March 2008
Line 11:		Line 11:

	*2-4 μL each purified, linearized DNA		*2-4 μL each purified, linearized DNA
-	*~~1 μL 10X~~ Ligase ~~Buffer~~	+	*1X Ligase buffer
	*200 units T4 DNA Ligase		*200 units T4 DNA Ligase
	deionized H<sub>2</sub>O to 10μL		deionized H<sub>2</sub>O to 10μL

Reshma P. Shetty at 03:40, 18 March 2008

2008-03-18T03:40:05Z

←Older revision		Revision as of 03:40, 18 March 2008
Line 17:		Line 17:
	==Procedure==		==Procedure==

-	#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube	+	#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube.
	#Add ligation buffer. <br>Vortex buffer before pipetting to ensure that it is well-mixed. <br>Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. It is recommended that you aliquot the Ligation Buffer into smaller quantities.		#Add ligation buffer. <br>Vortex buffer before pipetting to ensure that it is well-mixed. <br>Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. It is recommended that you aliquot the Ligation Buffer into smaller quantities.
	#Add prefix part, suffix part and destination vector to the tube.		#Add prefix part, suffix part and destination vector to the tube.

Reshma P. Shetty at 03:36, 18 March 2008

2008-03-18T03:36:32Z

Reshma P. Shetty: New page: ==Materials== [http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA ligase] from [http://www.neb.com/ NEB] Deionized, sterile H₂O *Purified, linearized destination ve...

2008-03-18T03:27:27Z

New page: ==Materials== *[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA ligase] from [http://www.neb.com/ NEB] *Deionized, sterile H2O *Purified, linearized destination ve...

New page

==Materials==

*[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA ligase] from [http://www.neb.com/ NEB]
*Deionized, sterile H2O
*Purified, linearized destination vector (in H2O)
*Purified, linearized prefix part (in H2O)
*Purified, linearized suffix part (in H2O)

==Ligation Mix==

*2-4 μL each purified, linearized DNA
*1 μL 10X Ligase Buffer
*200 units T4 DNA Ligase
deionized H2O to 10μL

==Procedure==

#Add appropriate amount of deionized H2O to sterile 0.6 mL tube
#Add 1 μL ligation buffer to the tube. Vortex buffer before pipetting to ensure that it is well-mixed. Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. It is recommended that you aliquot the Ligation Buffer into smaller quantities.
#Add prefix part, suffix part and destination vector to the tube.
#Add T4 DNA ligase. Vortex ligase before pipetting to ensure that it is well-mixed. Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add the correct amount, just touch your tip to the surface of the liquid when pipetting.
#Incubate 20 minutes on the benchtop at room-temperature.
#Place on ice until transformation.

[[Category:DNA]] [[Category:In vitro]] [[Category:Protocol]]

←Older revision		Revision as of 03:36, 18 March 2008
Line 1:		Line 1:
	==Materials==		==Materials==

-	*[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA ~~ligase~~] from [http://www.neb.com/ NEB]	+	*[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA Ligase] from [http://www.neb.com/ NEB]
		+	*10X T4 DNA Ligase buffer
	*Deionized, sterile H<sub>2</sub>O		*Deionized, sterile H<sub>2</sub>O
	*Purified, linearized destination vector (in H<sub>2</sub>O)		*Purified, linearized destination vector (in H<sub>2</sub>O)
Line 17:		Line 18:

	#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube		#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube
-	#Add ~~1 μL~~ ligation buffer ~~to the tube~~. <br>Vortex buffer before pipetting to ensure that it is well-mixed. <br>Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. It is recommended that you aliquot the Ligation Buffer into smaller quantities.	+	#Add ligation buffer. <br>Vortex buffer before pipetting to ensure that it is well-mixed. <br>Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. It is recommended that you aliquot the Ligation Buffer into smaller quantities.
	#Add prefix part, suffix part and destination vector to the tube.		#Add prefix part, suffix part and destination vector to the tube.
-	#Add T4 DNA ~~ligase~~. <br>Vortex ligase before pipetting to ensure that it is well-mixed. <br>Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add the correct amount, just touch your tip to the surface of the liquid when pipetting.	+	#Add T4 DNA Ligase. <br>Vortex ligase before pipetting to ensure that it is well-mixed. <br>Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add the correct amount, just touch your tip to the surface of the liquid when pipetting.
	#Incubate 20 minutes on the benchtop at room-temperature.		#Incubate 20 minutes on the benchtop at room-temperature.
	#Place on ice until transformation.		#Place on ice until transformation.

	[[Category:DNA]] [[Category:In vitro]] [[Category:Protocol]]		[[Category:DNA]] [[Category:In vitro]] [[Category:Protocol]]

Engineering BioBrick vectors from BioBrick parts/DNA ligation - Revision history

Reshma P. Shetty at 15:42, 18 March 2008

Reshma P. Shetty at 15:36, 18 March 2008

Reshma P. Shetty at 15:36, 18 March 2008

Reshma P. Shetty at 15:18, 18 March 2008

Reshma P. Shetty at 15:18, 18 March 2008

Reshma P. Shetty at 03:40, 18 March 2008

Reshma P. Shetty at 03:36, 18 March 2008

Reshma P. Shetty: New page: ==Materials== *[http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA ligase] from [http://www.neb.com/ NEB] *Deionized, sterile H2O *Purified, linearized destination ve...

Reshma P. Shetty: New page: ==Materials== [http://www.neb.com/nebecomm/products/productM0202.asp T4 DNA ligase] from [http://www.neb.com/ NEB] Deionized, sterile H₂O *Purified, linearized destination ve...