Griffitts:Restriction digest

Restriction Digest

Materials

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

Recipes

15 μL reaction

• 5 μL DNA
• 1.5 μL appropriate 10X buffer
• 1.5 μL 10X BSA (if needed—see below)
• 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 15 μL

Note: Use this recipe when verifying plasmid inserts
Note: You can check this reaction after 1 hour

30 μL reaction

• 12 μL DNA
• 3.0 μL appropriate 10X buffer
• 3.0 μL 10X BSA (if needed—see below)
• 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 30 μL

Note: Use this recipe when your DNA concentrations are sufficient
Note: For a BamHI–XbaI double-digest, use 3.3 μL of 10X buffer

40 μL reaction

• 20 μL DNA
• 4.0 μL appropriate 10X buffer
• 4.0 μL 10X BSA (if needed—see below)
• 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

Note: Use this recipe when your DNA concentrations are low
Note: For a BamHI–XbaI double-digest, use 4.3 μL of 10X buffer

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 or proceed to in-gel ligation

Enzymes

Restriction Enzymes

Enzyme	Amount	Target	Overhang	NEBuffer1	NEBuffer2	NEBuffer3	NEBuffer4	Other Buffer	T°	BSA
Avr II		c/ctagg	ctag	100	1001	50	100	None	37°C	No
Bam HI	0.8 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	100	None	37°C	Yes
Xho I		c/tcgag	tcga	75	100	100	100	None	37°C	Yes

¹Boldface indicates the preferred buffer for the enzyme.

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
Bam HI	seq1	-	-	-	-	-	-	-	-	-	-	-	-	-	-
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	-	-	-	-	-	-	-	-	-
Nde I	4	Bam HI	3	Eco RI	2	2	1	-	-	-	-	-	-	-	-
Pst I	3	Bam HI	3	Eco RI	3	2	1	3	-	-	-	-	-	-	-
Sac I	1	seq	2	seq	2	2	1	4	1	-	-	-	-	-	-
Sal I	seq	Bam HI	3	Eco RI	3	seq	seq	3	3	seq	-	-	-	-	-
Sma I	4	seq	seq	seq	4	4	seq	4	4	4	seq	-	-	-	-
Spe I	2	Bam HI	2	Eco RI	2	2	1	2	2	1	seq	4	-	-	-
Sph I	2	Bam HI	2	Eco RI	2	2	1	2	2	1	seq	4	2	-	-
Xba I	2	seq2	2	2	2	2	2	2	3	4	3	4	2	2	-
Xho I	2	Bam HI	3	Eco RI	3	2	1	4	3	1	3	4	2	2	2

¹Sequential digest recommended.
²We actually prefer to do the double-digest and use Buffer 2 for the BamHI–XbaI double-digest.
Note that a double-digest of Bam HI and Xba I is not recommended. However, by following the procedure above you won't have any problems.

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