Knight:TOPO vector preparation

Overview

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

Materials

• pSB**5 vectors
• Nt.BstNB I nicking enzyme
• Topoisomerase I from Vaccinia

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 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 Incubate at 37°C for at least 1 hour

Unit test definition 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 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?