Proteinase K Protocol: Difference between revisions
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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. | The following protocol is for a total volume of 10mL Proteinase K solution with a final concentration of 1 mg/mL. | ||
5mL Water | ==Materials== | ||
5mL 100% Glycerol (final concentration 50%) | |||
22mg CaCl2 anhydrous (M.W. = 110.99 g/mol) | *5mL Water | ||
16mg Tris-HCl anhydrous (M.W. = 121.14 g/mol) | *5mL 100% Glycerol (final concentration 50%) | ||
10mg proteinase K | *22mg CaCl2 anhydrous (M.W. = 110.99 g/mol) | ||
*16mg Tris-HCl anhydrous (M.W. = 121.14 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. | 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 | First, the buffer is needed. The following masses are only applicable for a final volume of 10mL | ||
For 20mM CaCl2: | For 20mM CaCl2: | ||
- 20 mmol/L = 2x10^-5 mol/mL | - 20 mmol/L = 2x10^-5 mol/mL | ||
- 2x10^-5 mol/mL * 110.99 g/mol * 10mL = 22.2mg CaCl2 | - 2x10^-5 mol/mL * 110.99 g/mol * 10mL = 22.2mg CaCl2 | ||
For 10 mM Tris-HCl: | For 10 mM Tris-HCl: | ||
- 10 mmol/L = 1x10^-5 mol/mL | - 10 mmol/L = 1x10^-5 mol/mL | ||
- 1x10^-5 mol/mL * 121.14 g/mol * 10mL = 15.8mg Tris-HCl | - 1x10^-5 mol/mL * 121.14 g/mol * 10mL = 15.8mg Tris-HCl | ||
Since the resolution of the scale is 1mg, we will measure 22mg CaCl2 and 16mg 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. | 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. | 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. |
Latest revision as of 00:03, 8 April 2010
The following protocol is for a total volume of 10mL Proteinase K solution with a final concentration of 1 mg/mL.
Materials[edit]
- 5mL Water
- 5mL 100% Glycerol (final concentration 50%)
- 22mg CaCl2 anhydrous (M.W. = 110.99 g/mol)
- 16mg Tris-HCl anhydrous (M.W. = 121.14 g/mol)
- 10mg proteinase K
Calculations[edit]
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 * 121.14 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[edit]
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.