Griffitts:Restriction digest

Home     Research     Lab Members     Protocols     Publications     Internal     Emeritus    

Restriction Digest

Materials

• ddH₂0
• DNA sample on ice
• Appropriate 10X buffer on ice
• 10X BSA on ice
• Restriction enzyme(s) (see below) on ice
• CIP (keep in freezer until needed)

Recipe

• 12-20 μL DNA depending on concentration
• 4.3 μL appropriate 10X buffer
• 4.0 μL 10X BSA
• Restriction enzyme(s) (see specific enzymes below for exact amounts)
  • Don't immerse the tip, draw from the surface to avoid excess enzyme
• Add ddH₂0 to bring the final volume to 40 μL

Procedure

• Combine ingredients for recipe
• Incubate at 37°C for 2.5 to 8 hours
• If preparing a ligation, add 0.5 μL CIP to the vector 30 minutes prior to ending the reaction
• When the reaction is complete you can store your samples in the -20°C freezer

Enzymes

Restriction Enzymes

Enzyme	Amount	Target	Overhang	NEBuffer1	NEBuffer2	NEBuffer3	NEBuffer4	Other Buffer	T°	BSA
Avr II		c/ctagg	ctag	100	100	50	100	None	37°C	No
Bam HI	1 μL	g/gatcc	gatc	75	100	50	75	Bam HI	37°C	Yes
Bgl II		a/gatct	gatc	10	75	100	10	None	37°C	No
Bst XI		ccannnnn/ntgg	nnnn	25	100	100	50	None	55°C	No
Eco RI		g/aattc	aatt	100	100	100	100	Eco RI	37°C	No
Eco RV		gat/atc	none	50	75	100	50	None	37°C	Yes
Hind III		a/agctt	agct	50	100	10	50	None	37°C	No
Kpn I		ggtac/c	gtac	100	75	0	75	None	37°C	Yes
Nde I		ca/tatg	ta	75	100	75	100	None	37°C	No
Pst I		ctgca/g	tgca	75	75	100	50	None	37°C	Yes
Sac I		gagct/c	agct	100	50	10	100	None	37°C	Yes
Sal I		g/tcgac	tcga	0	0	100	0	None	37°C	Yes
Sma I		ccc/ggg	none	0	0	0	100	None	25°C	No
Spe I		a/ctagt	ctag	75	100	25	75	None	37°C	Yes
Sph I		gcatg/c	catg	100	100	50	100	None	37°C	No
Xba I	1.5 μL	t/ctaga	ctag	0	100	75	75	None	37°C	Yes
Xho I		c/tcgag	tcga	75	100	100	100	None	37°C	Yes

Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.

Double Digests

Suggested Buffers

Enzyme	Avr II	Bam HI	Bgl II	Eco RI	Eco RV	Hind III	Kpn I	Nde I	Pst I	Sac I	Sal I	Sma I	Spe I	Sph I	Xba I	Xho I
Bam HI	seq	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Bgl II	3	Bam HI	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Eco RI	Eco RI	Eco RI	Eco RI	-	-	-	-	-	-	-	-	-	-	-	-	-
Eco RV	2	Bam HI	3	Eco RI	-	-	-	-	-	-	-	-	-	-	-	-
Hind III	2	seq	2	Eco RI	2	-	-	-	-	-	-	-	-	-	-	-
Kpn I	1	seq	2	2	2	2	-	-	-	-	-	-	-	-	-	-

Adapted from New England BioLabs 2005-06 Catalog and Technical Reference.