MIT iGEM Restriction Digests - Revision history

Felix Moser: /* Method */

2008-06-24T17:05:47Z

Method

←Older revision		Revision as of 17:05, 24 June 2008
Line 19:		Line 19:
	#Incubate the reaction at 37*C for 2hrs to ensure complete digestion.		#Incubate the reaction at 37*C for 2hrs to ensure complete digestion.
	#Deactivate the enzymes by heating @ 80*C for 20min (in the thermocycler). This will inactivate most enzymes, though check to make sure that your enzyme can be heat deactivated by checking in the NEB catalogue.		#Deactivate the enzymes by heating @ 80*C for 20min (in the thermocycler). This will inactivate most enzymes, though check to make sure that your enzyme can be heat deactivated by checking in the NEB catalogue.
-	#~~Keep~~ digest at -20*C or run immediately on gel.	+	#Store digest at -20*C or run immediately on gel.

Felix Moser: /* Method */

2008-06-24T17:05:17Z

Method

←Older revision		Revision as of 17:05, 24 June 2008
Line 18:		Line 18:
	#MIX THE REACTION BY PIPETTING HALF THE VOLUME UP AND DOWN.		#MIX THE REACTION BY PIPETTING HALF THE VOLUME UP AND DOWN.
	#Incubate the reaction at 37*C for 2hrs to ensure complete digestion.		#Incubate the reaction at 37*C for 2hrs to ensure complete digestion.
-	#Deactivate the enzymes by heating ~~for~~ 80*C for 20min (in the thermocycler). This will inactivate most enzymes, though check to make sure that your enzyme can be heat deactivated by checking in the NEB catalogue.	+	#Deactivate the enzymes by heating @ 80*C for 20min (in the thermocycler). This will inactivate most enzymes, though check to make sure that your enzyme can be heat deactivated by checking in the NEB catalogue.
	#Keep digest at -20*C or run immediately on gel.		#Keep digest at -20*C or run immediately on gel.

Felix Moser: /* Materials */

2008-06-24T17:02:29Z

Materials

←Older revision		Revision as of 17:02, 24 June 2008
Line 3:		Line 3:

	==Materials==		==Materials==
-	*DNA; the thing you want to cut.	+	*DNA; the thing you want to cut. Usually plasmid or PCR product. Measure concentration in Nanodrop beforehand.
	*Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers).		*Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers).
	*Appropriate enzymes.		*Appropriate enzymes.

Felix Moser: /* Restriction Digest protocol */

2008-06-24T16:58:57Z

Restriction Digest protocol

←Older revision		Revision as of 16:58, 24 June 2008
Line 1:		Line 1:
	=Restriction Digest protocol=		=Restriction Digest protocol=
		+	'''For Analytical digests, aim for 500-1000ng of DNA; for a preparative digest (where you know you want to cut out a band) try to load as much DNA as possible (gel lanes w/ wide-toothed combs can hold up to 50µl).'''
		+
	==Materials==		==Materials==
	*DNA; the thing you want to cut.		*DNA; the thing you want to cut.

Felix Moser: /* Method */

2008-06-24T16:51:33Z

Method

←Older revision		Revision as of 16:51, 24 June 2008
Line 8:		Line 8:

	==Method==		==Method==
-	'''The following volumes apply to a 20µl analytical digest; for larger, preparative digests, simply scale up (eg. for a 30µl digest, use 3µl of 10x buffer'''	+	'''The following volumes apply to a 20µl analytical digest; for larger, preparative digests, simply scale up (eg. for a 30µl digest, use 3µl of 10x buffer, etc)'''
	#Add 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube. (eg. 20µl-(1µlDNA+2µlBuffer+0.2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=15.8µl ddH2O)		#Add 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube. (eg. 20µl-(1µlDNA+2µlBuffer+0.2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=15.8µl ddH2O)
	#Add 0.2µl BSA to tube.		#Add 0.2µl BSA to tube.

Felix Moser: /* Method */

2008-06-24T16:51:14Z

Method

←Older revision		Revision as of 16:51, 24 June 2008
Line 8:		Line 8:

	==Method==		==Method==
-	'''The following ~~is for~~ a 20µl analytical digest; for larger, preparative digests, simply scale up'''	+	'''The following volumes apply to a 20µl analytical digest; for larger, preparative digests, simply scale up (eg. for a 30µl digest, use 3µl of 10x buffer'''
-	#Add 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube.	+	#Add 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube. (eg. 20µl-(1µlDNA+2µlBuffer+0.2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=15.8µl ddH2O)
	#Add 0.2µl BSA to tube.		#Add 0.2µl BSA to tube.
	#Add 2.0µl 10x Buffer to tube.		#Add 2.0µl 10x Buffer to tube.

Felix Moser: /* Materials */

2008-06-24T16:47:29Z

Materials

←Older revision		Revision as of 16:47, 24 June 2008
Line 5:		Line 5:
	*Appropriate enzymes.		*Appropriate enzymes.
	*ddH2O		*ddH2O
-	*BSA (~~10,000x~~ from NEB)	+	*BSA (100x from NEB)

	==Method==		==Method==

Felix Moser: New page: =Restriction Digest protocol= ==Materials== DNA; the thing you want to cut. Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers). *Appropriate...

2008-06-24T16:46:37Z

New page: =Restriction Digest protocol= ==Materials== *DNA; the thing you want to cut. *Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers). *Appropriate...

New page

=Restriction Digest protocol=
==Materials==
*DNA; the thing you want to cut.
*Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers).
*Appropriate enzymes.
*ddH2O
*BSA (10,000x from NEB)

==Method==
'''The following is for a 20µl analytical digest; for larger, preparative digests, simply scale up'''
#Add 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube.
#Add 0.2µl BSA to tube.
#Add 2.0µl 10x Buffer to tube.
#Add appropriate amount of DNA to tube.
#Add 0.5µl of each enzyme to tube.
#MIX THE REACTION BY PIPETTING HALF THE VOLUME UP AND DOWN.
#Incubate the reaction at 37*C for 2hrs to ensure complete digestion.
#Deactivate the enzymes by heating for 80*C for 20min (in the thermocycler). This will inactivate most enzymes, though check to make sure that your enzyme can be heat deactivated by checking in the NEB catalogue.
#Keep digest at -20*C or run immediately on gel.

MIT iGEM Restriction Digests - Revision history

Felix Moser: /* Method */

Felix Moser: /* Method */

Felix Moser: /* Materials */

Felix Moser: /* Restriction Digest protocol */

Felix Moser: /* Method */

Felix Moser: /* Method */

Felix Moser: /* Materials */

Felix Moser: New page: =Restriction Digest protocol= ==Materials== *DNA; the thing you want to cut. *Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers). *Appropriate...

Felix Moser: New page: =Restriction Digest protocol= ==Materials== DNA; the thing you want to cut. Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers). *Appropriate...