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.
[edit]Materials


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)
[edit]Calculations
*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.
[edit]Procedure
 
==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.