Knight:TOPO vector preparation: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
Line 29: | Line 29: | ||
Do a purification step? Or use it directly? | Do a purification step? Or use it directly? | ||
===ATP?=== | |||
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 MgCl<sub>2</sub> | |||
*5 mM ATP | |||
*0.3 μg of plasmid DNA | |||
*0.63ng of ''Vaccinia'' topoisomerase I, incubated at 37°C for five minutes. | |||
On 7/19, I tried the above mixture, except I used 5 μL DNA and 1 μL enzyme. Also, the incubation time was for fifteen minutes instead of five. [[User:Yeem|yeem]] 15:31, 19 July 2007 (EDT) |
Revision as of 07:55, 25 July 2007
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
- 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
- Mix
- 50 mM Tris-acetate (pH7.5)
- 100mM 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
Do a purification step? Or use it directly?
ATP?
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.
On 7/19, I tried the above mixture, except I used 5 μL DNA and 1 μL enzyme. Also, the incubation time was for fifteen minutes instead of five. yeem 15:31, 19 July 2007 (EDT)