Knight:Protein DNA binding/Option 1

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.

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.

4M KCl

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.

5M potassium acetate

(different from Potassium Acetate)

To make 200mL

• Mix 98.15 g potassium acetate with 50 ml deionized water.
• Adjust volume to 200 ml with deionized water.

50% glycerol

To make 100mL

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

10% NP-40

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

• Mix 10mL NP40 with 90mL H₂O.

20mM zinc sulfate

To make 100mL

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

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.