New England Biolabs provides a color-coded 10X NEBuffer with every restriction endonuclease to ensure 100% activity. Some enzymes require an NEBuffer which is different from the four standard NEBuffers (1, 2, 3 or 4) for optimal activity. To help select the best conditions for double digests with these enzymes that require a unique (designated “U”) NEBuffer, the table below lists the activity of the most commonly used restriction endonucleases in the unique NEBuffers supplied with the following enzymes: BsrFI, DpnII, EcoRI and SspI.

Performing double digests with enzymes supplied with a unique NEBuffer and not one of the four standard NEBuffers is usually simple. In most cases, double digests using any of these enzymes with an enzyme that is supplied with a standard NEBuffers can be done in the unique NEBuffer. This will ensure that the enzyme with the more specific buffer requirements will work optimally. When using restriction endonucleases in non-optimal NEBuffers, more enzyme or longer digestion time may be needed to compensate for the slower rate of cleavage under those conditions.

If BSA or SAM is supplied with an enzyme, it is included in all NEBuffer activity reactions.

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NEBuffer Activity  | Double Digestion | Time-Saver Enzymes

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Enzyme		Supplied NEBuffer	% Activity in NEBuffers
			BsrFI	DpnII	EcoRI	SspI
AatII		NEBuffer 4	25	0	25	25
AvrII		NEBuffer 4	100	100	100	100
BamHI		NEBuffer 3+BSA	100	100	100	100
BglII		NEBuffer 3	50	0	50	50
BsgI		NEBuffer 4+SAM	10	10	10	10
EagI		NEBuffer 3	100	10	100	100
EcoRI		NEBuffer EcoRI	100	0	100dd	100
EcoRV		NEBuffer 3+BSA	100	0	100	100
HindIII		NEBuffer 2	50	0	50	50
KpnI		NEBuffer 1+BSA	25	10	25	25
MseI		NEBuffer 4+BSA	50	10	50	50
NcoI		NEBuffer 3	100	10	100	100
NdeI		NEBuffer 4	100	0	100	100
NheI		NEBuffer 2+BSA	75	50	75	75
NotI		NEBuffer 3+BSA	100	0	100	100
PstI		NEBuffer 3+BSA	50	0	50	50
PvuI		NEBuffer 3+BSA	75	0	75	75
SacI		NEBuffer 1+BSA	25	0	25	25
SacII		NEBuffer 4	100	0	100	100
SalI		NEBuffer 3+BSA	100	0	100	100
SmaI @25°C		NEBuffer 4	0	0	0	0
SpeI		NEBuffer 4+BSA	75	25	75	75
SphI		NEBuffer 2	50	10	50	50
XbaI		NEBuffer 4+BSA	10	10	10	10
XhoI		NEBuffer 4+BSA	100	0	100	100
XmnI		NEBuffer 4+BSA	10	0	10	10

Note: The values listed in this table are approximate. They were obtained using each enzyme's specific unit assay substrate DNA.