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Methods:DNA Knot Ladder

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Contents

Purpose

Colloms et al[1]used DNA Knot ladders as gel standards when looking at Xer recombination on plasmid cer and psi sites

Materials Needed

You will need:

  • negatively supercoiled DNA
  • S. cerevisiae Topoisomerase II

Reactions are done in

  • 50mM Tris- HCl (pH 7.5)
  • 120mM KCl
  • 6mM MgCl,sub>2</sub>
  • 1mM DTT
  • 200µg/ml BSA
  • 10µM ATP

Protocol

  • Incubate reactions containing 20µg/ml DNA and 30µg/ml Topo II for 4 minutes at 37C
  • Stop reaction with EDTA (doesn't specify as to how much)
  • Phenol extract DNA
  • EtOH precipitate DNA
  • DNase I treat as described in Methods:DNase I nicking

Links

User notes

References

See Help:References for how to manage references in EcoliWiki.

  1. Colloms, SD et al. (1997) Topological selectivity in Xer site-specific recombination. Cell 88 855-64 PubMed EcoliWiki page