Knight:Protein DNA binding/Option 3

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Contents

Stock solutions

500mM HEPES-NaOH pH 7.9

To make 200mL

  • Dissolve 23.83 g HEPES into 60mL H2O.
  • Adjust pH to 7.9 with 5N NaOH (use 6N NaOH if available).
  • Adjust volume to 200 mL with H2O.
  • Filter sterilize.

20mg/mL BSA

  • Dilute 1g BSA in 50mL DI water.
  • Filter sterilize

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.

20mM zinc sulfate

To make 100mL

  • Dissolve 0.575 g zinc sulphate heptahydrate in 60mL H2O.
  • Adjust volume to 100 ml with deionized water.
  • Filter sterilize? (I did.)

Making binding reaction buffer

To make 1mL

  • 200 μL 50% glycerol
  • 30 μL 500mM HEPES-NaOH
  • 25 μL 4M potassium chloride
  • 5 μL 1M magnesium chloride
  • 5 μL 20mM zinc sulfate
  • 5 μL 20mg/mL BSA

To make 10mL

  • 2 mL 50% glycerol
  • 300 μL 500mM HEPES-NaOH
  • 250 μL 4M potassium chloride
  • 50 μL 1M magnesium chloride
  • 50 μL 20mM zinc sulfate
  • 50 μL 20mg/mL BSA

To make 100mL

  • 20 mL 50% glycerol
  • 3 mL 500mM HEPES-NaOH
  • 2.5 mL 4M potassium chloride
  • 500 μL 1M magnesium chloride
  • 500 μL 20mM zinc sulfate
  • 500 μL 20mg/mL BSA


Add β-mercaptoethanol just before use (0.7 μL per mL of buffer).

Or add TCEP to final concentration of 1mM (2μL of 0.5M stock per mL of buffer).

Binding reaction conditions

  • 15mM Hepes-NaOH (pH 7.9)
    • Hepes is a better buffer than Tris
  • 100mM potassium chloride
  • 5 mM magnesium chloride
  • 100μM zinc sulfate
    • zinc sulfate goes into solution better than zinc chloride
    • higher zinc concentration to compensate for possible presence of reducing agents
  • 10mM β-mercaptoethanol --> Or add TCEP to final concentration of 1mM?
    • β-mercaptoethanol is a reducing agent that chelates zinc less tightly than DTT
  • 10% (v/v) glycerol
  • 100 μg/mL BSA
  • may want to titrate in different amounts of poly dI-dC
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