From OpenWetWare
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
- Mix
- X μL plasmid
- 1 μL Nt.BstNB I
- 5 μL NEBuffer 3
- 44-X μL H2O
- Incubate at 55°C for at least 1 hour
- 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
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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
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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.
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Do a purification step? Or use it directly?
