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Overview

This is a made-up protocol to prepare pSB**5 vectors for TOPO cloning. It has not been verified at all!

Materials

Procedure

Nick plasmid

  1. Mix
    • X μL plasmid
    • 1 μL Nt.BstNB I
    • 5 μL NEBuffer 3
    • 44-X μL H2O
  2. Incubate at 55°C for at least 1 hour
  3. Heat-inactivate at 80°C for 20 mins

Do a purification step? Try the same buffer?

Covalently attach DNA topoisomerase I from Vaccinia

Quality control procedure

  1. Mix a 50 μL reaction including
    • 50 mM Tris-acetate (pH7.5)
    • 100 mM NaCl
    • 2.5 mM MgCl2
    • 0.1 mM EDTA
    • 1 μL DNA topoisomerase I from Vaccinia
  2. Incubate at 37°C for at least 1 hour

Unit test definition

  1. Mix
    • 40 mM Tris-acetate (pH 8.3)
    • 100 mM NaCl
    • 2.5 mM MgCl2
    • 1 mM EDTA
    • 1 unit of DNA topoisomerase I from Vaccinia
  2. Incubate at 37°C for 1 hour

Alternative protocol from Shuman et al.

From Shuman 1994: maximum topoisomerase activity was observed with a 20 μL reaction mixture containing

  • 50 mM Tris-HCl (pH 8.0)
  • 100 mM NaCl
  • 5 mM MgCl2
  • 5 mM ATP
  • 0.3 μg of plasmid DNA
  • 0.63ng of Vaccinia topoisomerase I, incubated at 37°C for five minutes.

Do a purification step? Or use it directly?