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Restriction Enzyme Buffers

April 1, 1990

C. Helms


Enzymeconcentrationrecipe for 10XStore at
(source)in digest10X BufferBuffer (50 ml)degrees C
BamHI150 mM NaCl1.5 M NaCl15 ml 5 M NaCl -20 to +4 degrees C
(NE Biolabs)6 mM TrispH7.960 mM Tris-HCl3 ml 1 M Tris-HCl
6 mM MgCl260 mM MgCl23 ml 1 M MgCl2
100 g/ml BSA1 mg/ml BSA1 ml 50 mg/ml BSA
+dH2028 ml dH2O
BglII100 mM NaCl1.0 M NaCl10 ml 5 M NaCl -20 to +4 degrees C
(NE Biolabs)10 mM TrispH7.4100 mM Tris-HCl5 ml 1 M Tris-HCl
= HinfI buffer10 mM MgCl2100 mM MgCl25 ml 1 M MgCl2
= TaqI buffer100 g/ml BSA1 mg/ml BSA1 ml 50 mg/ml BSA
= PstI buffer+dH2029 ml dH2O
EcoRI50 mM NaCl0.5 M NaCl5 ml 5 M NaCl-20 to +4 degrees C
(NE Biolabs)100 mM TrispH7.50.82 M Tris-HCl 41.5 ml 1 M Tris-HCl
5 mM MgCl250 mM MgCl22.5 ml MgCl2
100 g/ml BSA1 mg/ml BSA1 ml 50 mg/ml BSA
HincII100 mM NaCl1 M NaCl10 ml 5 M NaCl +4 degrees C
(NE Biolabs)10 mM TrispH7.4100 mM Tris-HCl5 ml 1 M Tris-HCl
7 mM MgCl270 mM MgCl23.5 ml 1 M MgCl2
1 mM DTT10 mM DTT5 ml 100 mM DTT
100 g/ml BSA1 mg/ml BSA1 ml 50 mg/ml BSA
+dH2025.5 ml dH2O
HindIII10 mM Tris-HCl100 mM Tris-HCl5 ml 1 M Tris-HCl-20 degrees C
(MVO lab)50 mM NaCl0.5 M NaCl5 ml 5 M NaCl
10 mM MgCl2100 mM MgCl25 ml 1 M MgCl2
10 mM 2-ME100 mM 2-ME0.42 ml 12 M 2-ME
100 g/ml BSA1 mg/ml BSA1 ml 50 mg/ml BSA
+dH2033.58 ml dH2O
MspI6 mM KCl60 mM KCl3 ml 1 M KCl +4 degrees C
(NE Biolabs)10 mMTrispH7.4100 mM Tris-HCl5 ml 1 M Tris-HCl
10 mM MgCl2100 mM MgCl25 ml 1 M MgCl2
1 mM DTT10 mM DTT5 ml 100 mM DTT
100 g/ml BSA1 mg/ml BSA1 ml 50 mg/ml BSA
+dH2031 ml dH2O
RsaI50 mM NaCl0.5 M NaCl5 ml 5 M NaCl-20 degrees C
(NE Biolabs)6 mM TrispH8.060 mM Tris-HCl3 ml 1 M Tris-HCl
= AluI12 mM MgCl2120 mM MgCl26 ml 1 M MgCl2
= HaeIII6 mM 2-ME60 mM 2-ME0.26 ml 12M 2-ME
100 g/ml BSA1 mg/ml BSA1 ml 50 mg/ml BSA
+dH2034.74 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 l of the 12M 2-ME stock per ml will restore the buffer