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Restriction Enzyme Buffers

April 1, 1990

C. Helms

Enzyme	concentration		recipe for 10X	Store at
(source)	in digest	10X Buffer	Buffer (50 ml)	degrees C
BamHI	150 mM NaCl	1.5 M NaCl	15 ml 5 M NaCl	-20 to +4 degrees C
(NE Biolabs)	6 mM TrispH7.9	60 mM Tris-HCl	3 ml 1 M Tris-HCl
	6 mM MgCl2	60 mM MgCl2	3 ml 1 M MgCl2
	100 µg/ml BSA	1 mg/ml BSA	1 ml 50 mg/ml BSA
		+dH20	28 ml dH2O
BglII	100 mM NaCl	1.0 M NaCl	10 ml 5 M NaCl	-20 to +4 degrees C
(NE Biolabs)	10 mM TrispH7.4	100 mM Tris-HCl	5 ml 1 M Tris-HCl
= HinfI buffer	10 mM MgCl2	100 mM MgCl2	5 ml 1 M MgCl2
= TaqI buffer	100 µg/ml BSA	1 mg/ml BSA	1 ml 50 mg/ml BSA
= PstI buffer		+dH20	29 ml dH2O
EcoRI	50 mM NaCl	0.5 M NaCl	5 ml 5 M NaCl	-20 to +4 degrees C
(NE Biolabs)	100 mM TrispH7.5	0.82 M Tris-HCl	41.5 ml 1 M Tris-HCl
	5 mM MgCl2	50 mM MgCl2	2.5 ml MgCl2
	100 µg/ml BSA	1 mg/ml BSA	1 ml 50 mg/ml BSA
HincII	100 mM NaCl	1 M NaCl	10 ml 5 M NaCl	+4 degrees C
(NE Biolabs)	10 mM TrispH7.4	100 mM Tris-HCl	5 ml 1 M Tris-HCl
	7 mM MgCl2	70 mM MgCl2	3.5 ml 1 M MgCl2
	1 mM DTT	10 mM DTT	5 ml 100 mM DTT
	100 µg/ml BSA	1 mg/ml BSA	1 ml 50 mg/ml BSA
		+dH20	25.5 ml dH2O
HindIII	10 mM Tris-HCl	100 mM Tris-HCl	5 ml 1 M Tris-HCl	-20 degrees C
(MVO lab)	50 mM NaCl	0.5 M NaCl	5 ml 5 M NaCl
	10 mM MgCl2	100 mM MgCl2	5 ml 1 M MgCl2
	10 mM 2-ME	100 mM 2-ME	0.42 ml 12 M 2-ME
	100 µg/ml BSA	1 mg/ml BSA	1 ml 50 mg/ml BSA
		+dH20	33.58 ml dH2O
MspI	6 mM KCl	60 mM KCl	3 ml 1 M KCl	+4 degrees C
(NE Biolabs)	10 mMTrispH7.4	100 mM Tris-HCl	5 ml 1 M Tris-HCl
	10 mM MgCl2	100 mM MgCl2	5 ml 1 M MgCl2
	1 mM DTT	10 mM DTT	5 ml 100 mM DTT
	100 µg/ml BSA	1 mg/ml BSA	1 ml 50 mg/ml BSA
		+dH20	31 ml dH2O
RsaI	50 mM NaCl	0.5 M NaCl	5 ml 5 M NaCl	-20 degrees C
(NE Biolabs)	6 mM TrispH8.0	60 mM Tris-HCl	3 ml 1 M Tris-HCl
= AluI	12 mM MgCl2	120 mM MgCl2	6 ml 1 M MgCl2
= HaeIII	6 mM 2-ME	60 mM 2-ME	0.26 ml 12M 2-ME
	100 µg/ml BSA	1 mg/ml BSA	1 ml 50 mg/ml BSA
		+dH20	34.74 ml dH2O

Stock solutions for restriction enzyme buffers:

Always check the enzyme manufacturer's recommended buffer conditions before setting up digests. Prepare the 10X Buffers by combining all ingredients except the BSA. Filter sterilize, then add the sterile BSA. Prepare 1 ml aliquots, and store at the recommended temperatures. Buffers containing 2-ME can be stored in the refrigerator for a few weeks. If the mercaptoethanol is undetectible in refrigerated buffer, adding 6 µl of the 12M 2-ME stock per ml will restore the buffer

DTT (dithiothreitol), 100 mM stock:

Prepare in dH2O, filter sterilize and store at -20 degrees C

NaCl, 5 M stock:

Prepare in dH2O, filter sterilize, store at room temperature.

KCl, 1 M stock:

Prepare in dH2O, filter sterilize, store at room temperature.

Tris-HCl, pH 7.5- 8.3, 1 M stock:

Filter sterilize lab stock and store at room temperature.

MgCl2, 1 M stock:

Filter sterilize lab stock and store at room temperature.

2-mercaptoethanol (2-ME), 12 M stock:

Stored in the refrigerator. Pipet under the hood.

BSA (bovine serum albumin), 50 mg/ml stock:

Purchase from BRL, store at -20 degrees C.

100 mM Spermidine stock:

Weigh out 0.25 g spermidine tetrahydrochloride.
Add ddH2O to bring volume to 10 ml.
Filter sterilize and store at 4 degrees C.