Restriction Enzyme Buffer Most enzymes can use REact buffers; however, some are made up separately. Use fresh Milli-Q water, siliconized or sterile glassware or disposable plastic ware to make the following stock solutions up fresh (discard after use), combine to make 5 ml of 10X reaction buffers, put 1 ml in to each of 5 microfuge tubes (MFTs) and store the current one at -20C and the rest at -70C.
A: 1 M Tris-HCl 50 ml: 4.88 g Tris-HCl + 2.3 g Tris-Base (pH 7.9 at 25 C)
B: 1 M Tris-HCl 50 ml: 6.61 g Tris-HCl + 0.8 g Tris-Base (pH 7.4 at 25 C)
C: 1 M MgCl2 10 ml: 2.03 g
D: 3 M NaCl 20 ml: 3.50 g
E: 2M KCl 10 ml: 1.48 g
Ban I: 0.3ml B + 0.3ml C + 0.10ml D + 0.1ml BSA + 21μl bME + 4.18ml H2O
Bgl II: 0.5ml B + 0.5ml C + 1.67ml D + 0.1ml BSA + 35μl bME + 2.20ml H2O
Dra I: 0.5ml A + 0.5ml C + 0.83ml D + 0.1ml BSA + + 3.06ml H2O
Hpa II: 0.5ml B + 0.5ml C + 0.25ml E + 0.1ml BSA + 35μl bME + 3.62ml H2O
Nae I: 0.5ml A + 0.5ml C + 0.33ml D + 0.1ml BSA + 18μl bME + 3.55ml H2O
Not I: 0.5ml A + 0.5ml C + 2.50ml D + 0.1ml BSA + 5μl TX100+ 1.40ml H2O
Pst I: 0.5ml B + 0.5ml C + 1.67ml D + 0.1ml BSA + + 2.23ml H2O
Sal I: 0.5ml A + 0.5ml C + 2.50ml D + 0.1ml BSA + 35μl bME + 1.36ml H2O
Sca I/Ssp I: 2.5ml B + 0.3ml C + 0.83ml D + 0.1ml BSA + 35μl bME + 1.25ml E
Sfi I: 0.5ml A + 0.5ml C + 0.83ml D + 0.1ml BSA + 35μl bME + 3.04ml H2O
1X concentration:
Enzyme: [Tris-HCl]. [MgCl2], [NaCl]
Ban I: 6mM pH 7.4, 6mM, 6mM, 6mM bME
Bgl II: 10mM pH 7.4, 10mM, 100mM, 10mM bME
BstN I: 10mM pH 8.0, 10mM, 150mM
DraI: 10mM pH 8.0, 10mM, 50mM
Hpa II: 10mM pH 7.4, 10mM, 10mM bME, 10mM KCl
Mlu I: 10mM pH 7.5, 10mM, 50mM, 10mM bME,
Nae I: 10mM pH 8.0, 10mM, 20mM, 5mM bME
Nde I: 10mM pH 7.8, 7mM, 150mM, 6mM bME
Not I: 10mM pH 7.9, 10mM, 150mM, 0.01% Triton X
Nsi I: 10mM pH 8.4, 10mM, 150mM
Pst I: 10mM pH 7.5, 10mM, 100mM
PvuII: 10mM pH 7.5, 10mM, 60mM, 10mM bME
Sal I: 10mM pH 7.9, 10mM, 150mM, 10mM bME
Sca I: 50mM pH 7.9, 6mM, 50mM, 10mM bME, 50mM KCl
Sfi I: 10mM pH 7.8, 10mM, 50mM, 10mM bME
Ssp I: 50mM pH 7.9, 6mM, 50mM, 10mM bME, 50mM KCl