Knight:Protein DNA binding/Option 1: Difference between revisions

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MgCl<sub>2</sub> is very hygroscopic.  May want to order a fresh bottle since bottles should not be stored for a long time.
MgCl<sub>2</sub> is very hygroscopic.  May want to order a fresh bottle since bottles should not be stored for a long time.


===1M HEPES-NaOH pH 7.9===
===4M potassium chloride===
To make 100mL
*Dissolve 23.83 g HEPES into 60mL H<sub>2</sub>O.
*Adjust pH to 7.9 with 6N NaOH.
*Adjust volume to 100 mL with H<sub>2</sub>O.
*Filter sterilize.
 
===4M KCl===
To make 100mL
To make 100mL
*Dissolve 29.82 g solid KCl in 60 mL H<sub>2</sub>O.
*Dissolve 29.82 g solid KCl in 60 mL H<sub>2</sub>O.
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*Autoclave for 20 minutes on liquid cycle and store at room temperature.   
*Autoclave for 20 minutes on liquid cycle and store at room temperature.   
*Aliquot into 100 &mu;L aliquots in sterile tubes and use one at a time.
*Aliquot into 100 &mu;L aliquots in sterile tubes and use one at a time.
Note that this concentration of potassium chloride solution is near the solubility limits for potassium chloride in water.  So you may need to heat slightly to help the salt go into solution.


===5M potassium acetate===
===5M potassium acetate===
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To make 200mL
To make 200mL
*Mix 98.15 g potassium acetate with 50 ml deionized water.   
*Mix 98.14 g potassium acetate with 50 ml deionized water.   
*Adjust volume to 200 ml with deionized water.  
*Adjust volume to 200 ml with deionized water.  


===Potassium glutamate===
===20mM zinc sulfate===
To make 100mL
*Dissolve 0.575 g zinc sulphate heptahydrate in 60mL H<sub>2</sub>O.
*Adjust volume to 100 ml with deionized water.


===50% glycerol===
===50% glycerol===
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*Mix 50mL glycerol with 50mL H<sub>2</sub>O.
*Mix 50mL glycerol with 50mL H<sub>2</sub>O.
*Autoclave to sterilize.
*Autoclave to sterilize.
===1M HEPES-NaOH pH 7.9===
To make 100mL
*Dissolve 23.83 g HEPES into 60mL H<sub>2</sub>O.
*Adjust pH to 7.9 with 6N NaOH.
*Adjust volume to 100 mL with H<sub>2</sub>O.
*Filter sterilize.
===Potassium glutamate===


===10% NP-40===
===10% NP-40===
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*Mix 10mL NP40 with 90mL H<sub>2</sub>O.
*Mix 10mL NP40 with 90mL H<sub>2</sub>O.
===20mM zinc sulfate===
To make 100mL
*Dissolve 0.575 g zinc sulphate heptahydrate in 60mL H<sub>2</sub>O.
*Adjust volume to 100 ml with deionized water.


==Binding reaction conditions==
==Binding reaction conditions==

Revision as of 17:07, 11 September 2006

in progress! may contain errors.

Stock solutions

1M magnesium chloride

To make 100mL

  • Dissolve 20.33 g MgCl26H2O in 80mL H2O.
  • Adjust volume to 100 mL with H2O.
  • Dispense into aliquots and sterilize by autoclaving.

MgCl2 is very hygroscopic. May want to order a fresh bottle since bottles should not be stored for a long time.

4M potassium chloride

To make 100mL

  • Dissolve 29.82 g solid KCl in 60 mL H2O.
  • Adjust volume to 100 mL with H2O.
  • Autoclave for 20 minutes on liquid cycle and store at room temperature.
  • Aliquot into 100 μL aliquots in sterile tubes and use one at a time.

Note that this concentration of potassium chloride solution is near the solubility limits for potassium chloride in water. So you may need to heat slightly to help the salt go into solution.

5M potassium acetate

(different from Potassium Acetate)

To make 200mL

  • Mix 98.14 g potassium acetate with 50 ml deionized water.
  • Adjust volume to 200 ml with deionized water.

20mM zinc sulfate

To make 100mL

  • Dissolve 0.575 g zinc sulphate heptahydrate in 60mL H2O.
  • Adjust volume to 100 ml with deionized water.

50% glycerol

To make 100mL

  • Mix 50mL glycerol with 50mL H2O.
  • Autoclave to sterilize.

1M HEPES-NaOH pH 7.9

To make 100mL

  • Dissolve 23.83 g HEPES into 60mL H2O.
  • Adjust pH to 7.9 with 6N NaOH.
  • Adjust volume to 100 mL with H2O.
  • Filter sterilize.

Potassium glutamate

10% NP-40

Nonidet P40 (NP40). non-ionic surfactant-100mL. VWR catalog number 100304-536

  • Mix 10mL NP40 with 90mL H2O.

Binding reaction conditions

  • 15mM Hepes-NaOH (pH 7.9)
    • Hepes is a better buffer than Tris
  • 50mM KCl
    • it might be important that it is potassium salt rather than sodium
  • 50mM potassium glutamate
    • why this?
  • 50mM potassium acetate
    • why this?
  • 5mM MgCl2
    • this probably matters
  • 20μM ZnSO4
    • obviously need this
  • 2μg/mL BSA (from NEB)
    • acetylation is probably just to eliminate nuclease activity. Heat inactivation achieves the same result according to NEB.
  • 5% (v/v) glycerol
  • 0.1% (w/v) NP-40
    • detergent, likely important
  • 2 or 4 pM of the labeled site
    • need to calculate this and determine dilution series

10μL volume? Too small? Incubate 1hr at room temperature. Keep DNA concentration constant and vary protein amount.

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