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.

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.
• Autoclave to sterilize.

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.)

50% glycerol

To make 100mL

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

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.

1M potassium glutamate

• Dissolve 20.3236 g L-glutamic acid (monopotassium salt) in 60mL H₂O.
• Adjust volume to 100mL H₂O
• Filter sterilize? (I did.)

10% NP-40

• Nonidet P40 (NP40). non-ionic surfactant-100mL.
• VWR catalog number 100304-536.
• Obtained from Sauer lab. Store in dark at 2-4 °C.

20mg/mL BSA

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

(Don't use NEB BSA anymore because it has EDTA in it which might chelate the zinc even though it is present in very small quantities.)

Making binding reaction buffer

Or see the quick reference recipe.

To make 1mL ...

• 100μL 50% glycerol
• 50μL 1M potassium glutamate
• 30μL 500mM HEPES-NaOH (pH7.9)
• 10μL 10% NP-40
• 10μL 5M potassium acetate
• 12.5μL 4M potassium chloride
• 5μL 1M magnesium chloride
• 1μL 20mM zinc sulfate
• 2μL 1mg/mL BSA
• 779.5μL H₂O

To make 1000mL ...

• 100mL 50% glycerol
• 50mL 1M potassium glutamate
• 30mL 500mM HEPES-NaOH (pH7.9)
• 10mL 10% NP-40
• 10mL 5M potassium acetate
• 12.5mL 4M potassium chloride
• 5mL 1M magnesium chloride
• 1mL 20mM zinc sulfate
• 200μL 10mg/mL BSA
• 781.3mL H₂O

Binding reaction conditions

• 15mM Hepes-NaOH (pH 7.9)
  • Hepes is a better buffer than Tris
• 50mM potassium chloride
  • it might be important that it is potassium salt rather than sodium
• 50mM potassium glutamate
  • why this?
• 50mM potassium acetate
  • why this?
• 5mM magnesium chloride
  • this probably matters
• 20μM zinc sulfate
  • 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.

References

1. Greisman HA and Pabo CO. A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites. Science. 1997 Jan 31;275(5300):657-61. DOI:10.1126/science.275.5300.657 |
2. Wolfe SA, Ramm EI, and Pabo CO. Combining structure-based design with phage display to create new Cys(2)His(2) zinc finger dimers. Structure. 2000 Jul 15;8(7):739-50. DOI:10.1016/s0969-2126(00)00161-1 |