Engineering BioBrick vectors from BioBrick parts/Restriction digest - Revision history

Reshma P. Shetty at 15:41, 18 March 2008

Reshma P. Shetty — Tue, 18 Mar 2008 15:41:51 GMT

←Older revision		Revision as of 15:41, 18 March 2008
Line 5:		Line 5:
	*Deionized, sterile H<sub>2</sub>O		*Deionized, sterile H<sub>2</sub>O
	*0.5-1 μg DNA		*0.5-1 μg DNA
		+	*Sterile 0.6mL plastic tubes

	==Equipment==		==Equipment==

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Tue, 18 Mar 2008 15:36:25 GMT

Procedure

←Older revision		Revision as of 15:36, 18 March 2008
Line 19:		Line 19:

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

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Tue, 18 Mar 2008 15:35:19 GMT

Procedure

←Older revision		Revision as of 15:35, 18 March 2008
Line 19:		Line 19:

	==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
-	#Add restriction enzyme buffer~~. <br>Vortex buffer before pipetting to ensure that it is well-mixed~~.	+	#Add restriction enzyme buffer.
-	#Add BSA~~. <br>Vortex BSA before pipetting to ensure that it is well-mixed~~.	+	#Add BSA.
-	#Add DNA~~. <br>Vortex DNA before pipetting to ensure that it is well-mixed~~.	+	#Add DNA.
-	#Add each enzyme. ~~<br>Vortex enzyme before pipetting to ensure that it is well-mixed.~~ <br>Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.	+	#Add each enzyme. <br>Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.
	#Incubate for 2 hours at 37°C.		#Incubate for 2 hours at 37°C.
	#Incubate for 20 mins at 80°C to heat inactivate enzyme.<br> This step is sufficient to inactivate even Pst I.		#Incubate for 20 mins at 80°C to heat inactivate enzyme.<br> This step is sufficient to inactivate even Pst I.

Reshma P. Shetty at 15:22, 18 March 2008

Reshma P. Shetty — Tue, 18 Mar 2008 15:22:52 GMT

←Older revision		Revision as of 15:22, 18 March 2008
Line 5:		Line 5:
	*Deionized, sterile H<sub>2</sub>O		*Deionized, sterile H<sub>2</sub>O
	*0.5-1 μg DNA		*0.5-1 μg DNA
-	~~We performed all restriction digests by mixing 0.5-1 $\mu$g DNA, 1X NEBuffer 2, 100 $\mu$g/ml Bovine Serum Albumin, and 1 $\mu$L each needed restriction enzyme in a 50 $\mu$L total volume.~~

	==Equipment==		==Equipment==

Reshma P. Shetty: New page: ==Materials== *Restriction enzymes ([http://www.neb.com/nebecomm/products/productR0101.asp EcoRI], [http://www.neb.com/nebecomm/products/productR0133.asp SpeI], [http://www.neb.com/nebeco...

Reshma P. Shetty — Tue, 18 Mar 2008 15:22:41 GMT

New page: ==Materials== *Restriction enzymes ([http://www.neb.com/nebecomm/products/productR0101.asp EcoRI], [http://www.neb.com/nebecomm/products/productR0133.asp SpeI], [http://www.neb.com/nebeco...

New page

==Materials==

*Restriction enzymes ([http://www.neb.com/nebecomm/products/productR0101.asp EcoRI], [http://www.neb.com/nebecomm/products/productR0133.asp SpeI], [http://www.neb.com/nebecomm/products/productR0145.asp XbaI] or [http://www.neb.com/nebecomm/products/productR0140.asp PstI]) from [http://www.neb.com/ NEB]
*Bovine Serum Albumin (BSA)
*Deionized, sterile H2O
*0.5-1 μg DNA
We performed all restriction digests by mixing 0.5-1 $\mu$g DNA, 1X NEBuffer 2, 100 $\mu$g/ml Bovine Serum Albumin, and 1 $\mu$L each needed restriction enzyme in a 50 $\mu$L total volume.

==Equipment==

*DNA Engine Peltier Thermal Cycler (PTC-200) from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA).

==Digest mix==

*1X NEB2 buffer
*100 μg/mL BSA
*1 μL BioBrick enzyme 1
*1 μL BioBrick enzyme 2
deionized, sterile H2O to 50 μL

==Procedure==

#Add appropriate amount of deionized H2O to sterile 0.6 mL tube
#Add restriction enzyme buffer. Vortex buffer before pipetting to ensure that it is well-mixed.
#Add BSA. Vortex BSA before pipetting to ensure that it is well-mixed.
#Add DNA. Vortex DNA before pipetting to ensure that it is well-mixed.
#Add each enzyme. Vortex enzyme before pipetting to ensure that it is well-mixed. Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.
#Incubate for 2 hours at 37°C.
#Incubate for 20 mins at 80°C to heat inactivate enzyme. This step is sufficient to inactivate even Pst I.
#Incubate 4°C until you pull the reaction out of the thermal cycler.

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