Knight:Protein DNA binding/Option 2

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Contents

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. . pmid:10077584. PubMed HubMed [Segal-PNA-1999]
  2. Segal DJ, Crotty JW, Bhakta MS, Barbas CF 3rd, and Horton NC. . pmid:16963084. PubMed HubMed [Segal-JMB-2006]
All Medline abstracts: PubMed HubMed
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