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 -20℃ and the rest at -70℃. A: 1 M Tris-HCl 50 ml: 4.88 g Tris-HCl + 2.3 g Tris-Base (pH 7.9 at 25℃) B: 1 M Tris-HCl 50 ml: 6.61 g Tris-HCl + 0.8 g Tris-Base (pH 7.4 at 25℃) 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 Bg l II: 10mM pH 7.4, 10mM, 100mM, 10mM bME Bst N I: 10mM pH 8.0, 10mM, 150mM Dra I: 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 Pvu II: 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 |
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