Knight:Protein DNA binding/Option 2

Stock solutions

1M Tris-HCl pH7.6

Have a premade stock.

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.

Binding reaction conditions

• Zinc Buffer A
  • 10 mM Tris pH7.5
  • 90 mM KCl
  • 100 μM ZnCl₂ (or 90 μM ZnCl₂)
  • 1 mM MgCl₂
• 1% BSA
• 5 mM DTT
• 0.12 μg/μL sheared herring sperm DNA
• 10% glycerol
• 1 pM target oligonucleotide

References

1. Segal DJ, Dreier B, Beerli RR, and Barbas CF 3rd. Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2758-63. DOI:10.1073/pnas.96.6.2758 |
2. Segal DJ, Crotty JW, Bhakta MS, Barbas CF 3rd, and Horton NC. Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA. J Mol Biol. 2006 Oct 20;363(2):405-21. DOI:10.1016/j.jmb.2006.08.016 |