Knight:Restriction Digest - Revision history

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

Reshma P. Shetty — Tue, 18 Mar 2008 15:22:20 GMT

←Older revision		Revision as of 15:22, 18 March 2008
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	*Restriction enzymes ([http://www.neb.com/nebecomm/products/productR0101.asp EcoR I], [http://www.neb.com/nebecomm/products/productR0133.asp Spe I], [http://www.neb.com/nebecomm/products/productR0145.asp Xba I] or [http://www.neb.com/nebecomm/products/productR0140.asp Pst I]) from [http://www.neb.com/ NEB]		*Restriction enzymes ([http://www.neb.com/nebecomm/products/productR0101.asp EcoR I], [http://www.neb.com/nebecomm/products/productR0133.asp Spe I], [http://www.neb.com/nebecomm/products/productR0145.asp Xba I] or [http://www.neb.com/nebecomm/products/productR0140.asp Pst I]) from [http://www.neb.com/ NEB]
-	*~~EcoR I~~ buffer	+	*NEB2 buffer
	*BSA		*BSA
	*Deionized, sterile H<sub>2</sub>O		*Deionized, sterile H<sub>2</sub>O
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	##4°C forever (or until you pull the reaction out of the thermal cycler).		##4°C forever (or until you pull the reaction out of the thermal cycler).
	#Generally, use some method of [[Purification of DNA \| DNA purification]] to eliminate enzymes and salt from the reaction.		#Generally, use some method of [[Purification of DNA \| DNA purification]] to eliminate enzymes and salt from the reaction.
		+
		+
		+	[[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]]

Reshma P. Shetty: /* Digest Mix */

Reshma P. Shetty — Fri, 14 Dec 2007 01:13:09 GMT

Digest Mix

←Older revision		Revision as of 01:13, 14 December 2007
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	*5 μL NEB2 buffer (for all digests with BioBricks enzymes, we use NEB2 buffer. It keeps things simple and seems to work).		*5 μL NEB2 buffer (for all digests with BioBricks enzymes, we use NEB2 buffer. It keeps things simple and seems to work).
-	*X μL DNA (usually ~~~1 μg~~ depending on downstream uses).	+	*X μL DNA (usually ~500 ng depending on downstream uses).
	*0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest)		*0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest)
	*1 μL BioBricks enzyme 1 (regardless of the volume of the reaction, 1 μL enzyme is used because generally this represents a 10-25 fold excess of enzyme and is therefore sufficient for most digests. Also, it can be difficult to accurately pipet less than 1 μL of enzyme since it is sticky due to the glycerol content.)		*1 μL BioBricks enzyme 1 (regardless of the volume of the reaction, 1 μL enzyme is used because generally this represents a 10-25 fold excess of enzyme and is therefore sufficient for most digests. Also, it can be difficult to accurately pipet less than 1 μL of enzyme since it is sticky due to the glycerol content.)

Reshma P. Shetty at 23:58, 23 July 2007

Reshma P. Shetty — Mon, 23 Jul 2007 23:58:04 GMT

←Older revision		Revision as of 23:58, 23 July 2007
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	''Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.''		''Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.''

-	*5 μL ~~EcoR I~~ buffer (for all digests with BioBricks enzymes, we use ~~EcoR I~~ buffer. It keeps things simple and seems to work).	+	*5 μL NEB2 buffer (for all digests with BioBricks enzymes, we use NEB2 buffer. It keeps things simple and seems to work).
	*X μL DNA (usually ~1 μg depending on downstream uses).		*X μL DNA (usually ~1 μg depending on downstream uses).
	*0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest)		*0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest)

Reshma P. Shetty: /* Procedure */

Reshma P. Shetty — Mon, 09 Oct 2006 20:19:15 GMT

Procedure

←Older revision		Revision as of 20:19, 9 October 2006
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	#Add 1 μL of 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 1 μL of 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.
	#Place in thermal cycler ([http://www.mjr.com/ MJ Research], PT-200) and run digest protocol.		#Place in thermal cycler ([http://www.mjr.com/ MJ Research], PT-200) and run digest protocol.
-	##4-6 hour incubation at 37°C <br> Use a longer incubation time if you have time or are worried about the efficiency of cutting.	+	##4-6 hour incubation at 37°C <br> Use a longer incubation time if you have time or are worried about the efficiency of cutting. I think this time can be shortened to 2 hrs while still cutting to completion.
	##20 mins at 80°C to heat inactivate enzyme.<br> This step is sufficient to inactivate even Pst I.		##20 mins at 80°C to heat inactivate enzyme.<br> This step is sufficient to inactivate even Pst I.
	##4°C forever (or until you pull the reaction out of the thermal cycler).		##4°C forever (or until you pull the reaction out of the thermal cycler).
	#Generally, use some method of [[Purification of DNA \| DNA purification]] to eliminate enzymes and salt from the reaction.		#Generally, use some method of [[Purification of DNA \| DNA purification]] to eliminate enzymes and salt from the reaction.

Reshma P. Shetty at 03:34, 15 July 2005

Reshma P. Shetty — Fri, 15 Jul 2005 03:34:17 GMT

New page

==Materials==

*Restriction enzymes ([http://www.neb.com/nebecomm/products/productR0101.asp EcoR I], [http://www.neb.com/nebecomm/products/productR0133.asp Spe I], [http://www.neb.com/nebecomm/products/productR0145.asp Xba I] or [http://www.neb.com/nebecomm/products/productR0140.asp Pst I]) from [http://www.neb.com/ NEB]
*EcoR I buffer
*BSA
*Deionized, sterile H2O

==Digest Mix==

''Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.''

*5 μL EcoR I buffer (for all digests with BioBricks enzymes, we use EcoR I buffer. It keeps things simple and seems to work).
*X μL DNA (usually ~1 μg depending on downstream uses).
*0.5 μL 100X BSA (added to all digests because BSA never hurts a restriction digest)
*1 μL BioBricks enzyme 1 (regardless of the volume of the reaction, 1 μL enzyme is used because generally this represents a 10-25 fold excess of enzyme and is therefore sufficient for most digests. Also, it can be difficult to accurately pipet less than 1 μL of enzyme since it is sticky due to the glycerol content.)
*1 μL BioBricks enzyme 2
*(42.5 - X) μL deionized, sterile H2O

==Procedure==

#Add appropriate amount of deionized H2O to sterile 0.6 mL tube
#Add restriction enzyme buffer to the tube. Vortex buffer before pipetting to ensure that it is well-mixed.
#Add BSA to the tube. Vortex BSA before pipetting to ensure that it is well-mixed.
#Add appropriate amount of DNA to be cut to the tube. Vortex DNA before pipetting to ensure that it is well-mixed.
#Add 1 μL of 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.
#Place in thermal cycler ([http://www.mjr.com/ MJ Research], PT-200) and run digest protocol.
##4-6 hour incubation at 37°C Use a longer incubation time if you have time or are worried about the efficiency of cutting.
##20 mins at 80°C to heat inactivate enzyme. This step is sufficient to inactivate even Pst I.
##4°C forever (or until you pull the reaction out of the thermal cycler).
#Generally, use some method of [[Purification of DNA | DNA purification]] to eliminate enzymes and salt from the reaction.