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Proteinase K Preparation
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The following protocol is for a total volume of 10mL Proteinase K solution with a final concentration of 1 mg/mL. ==Materials== * 5mL Water * 5mL 100% Glycerol (final concentration 50%) * 22mg CaCl2 anhydrous (M.W. = 110.99 g/mol) * 16mg Tris-HCl anhydrous (M.W. = 157.60 g/mol) * 10mg proteinase K ==Calculations== The Proteinase K needs to be reconstituted in a solution of 10mM Tris-HCl, 20mM CaCl2, 50% Glycerol. We will consider a concentration of 1mg Proteinase K/mL solution to be 100% concentration. First, the buffer is needed. The following masses are only applicable for a final volume of 10mL For 20mM CaCl2: - 20 mmol/L = 2x10^-5 mol/mL - 2x10^-5 mol/mL * 110.99 g/mol * 10mL = 22.2mg CaCl2 For 10 mM Tris-HCl: - 10 mmol/L = 1x10^-5 mol/mL - 1x10^-5 mol/mL * 157.60 g/mol * 10mL = 15.8mg Tris-HCl Since the resolution of the scale is 1mg, we will measure 22mg CaCl2 and 16mg Tris-HCl. ==Procedure== First mix the 5mL water and 5mL 100% glyercol together. In no particular order, weigh out both the CaCl2 and Tris-HCl. Add one, mix until dissolved. Add the other and mix until dissolved. This is the buffer solution - 20mM CaCl2, 10mM Tris-HCl, 50% glycerol. To the buffer add 10mg Proteinase K for a concentration of 1 mg/mL. Any other concentrations can be easily calculated, as the total volume is 10mL.
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