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Methods:DNase I nicking

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Contents

Purpose

This method is used to release the constraints of supercoiling while keep the other topologies of substrate DNA (i.e. catenanes, knots, etc.)

Materials Needed

According to Colloms et al [1], the reaction mix is:

  • 50mM Tris-HCl (pH 8.0)
  • 10 mM MgCl2
  • 50 mM NaCl
  • 0.3 mg/ml EtBr
  • 1 µg/ml DNase I

According to Peng et al[2], the reaction mix is:

  • 40mM Tris-HCl (pH 7.6 at 30C)
  • 6 mM MgCl2
  • 20 mM KCl
  • 2 mM DTT
  • 1-8ng DNase I (various amounts were used in paper)

Protocol

According to Peng et al [2]:

Total reaction volume: 20µl
Incubation Time

  • 30C for 30 seconds

Termination of the reaction:

  • 20µl of stop solution

Product Recovery

  • Phenol extraction/ EtOH precipitation

Links

User notes

References

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  1. Colloms, SD et al. (1997) Topological selectivity in Xer site-specific recombination. Cell 88 855-64 PubMed EcoliWiki page
  2. 2.0 2.1 Peng, H & Marians, KJ (1995) The interaction of Escherichia coli topoisomerase IV with DNA. J. Biol. Chem. 270 25286-90 PubMed EcoliWiki page