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

Nick plasmid

 * Mix
 * 1) *X &mu;L plasmid
 * 2) *1 &mu;L Nt.BstNB I
 * 3) *5 &mu;L NEBuffer 3
 * 4) *44-X &mu;L H2O
 * 5) Incubate at 55&deg;C for at least 1 hour
 * 6) Heat-inactivate at 80&deg;C for 20 mins

Do a purification step? Try the same buffer?

Covalently attach DNA topoisomerase I from Vaccinia
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Quality control procedure

 * 1) Mix a 50 &mu;L reaction including
 * 2) *50 mM Tris-acetate (pH7.5)
 * 3) *100 mM NaCl
 * 4) *2.5 mM MgCl2
 * 5) *0.1 mM EDTA
 * 6) *1 &mu;L DNA topoisomerase I from Vaccinia
 * 7) Incubate at 37&deg;C for at least 1 hour

Unit test definition

 * Mix
 * 1) *40 mM Tris-acetate (pH 8.3)
 * 2) *100 mM NaCl
 * 3) *2.5 mM MgCl2
 * 4) *1 mM EDTA
 * 5) *1 unit of DNA topoisomerase I from Vaccinia
 * 6) Incubate at 37&deg;C for 1 hour

Alternative protocol from Shuman et al.
From Shuman 1994: maximum topoisomerase activity was observed with a 20 &mu;L reaction mixture containing
 * 50 mM Tris-HCl (pH 8.0)
 * 100 mM NaCl
 * 5 mM MgCl2
 * 5 mM ATP
 * 0.3 &mu;g of plasmid DNA
 * 0.63ng of Vaccinia topoisomerase I, incubated at 37&deg;C for five minutes.
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Do a purification step? Or use it directly?