Knight:TOPO vector preparation: Difference between revisions
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(New page: ==Overview== This is a made-up protocol to prepare pSB**5 vectors for TOPO cloning. It has not been verified at all! ==Materials== *[[Syntheti...) |
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#*5 μL NEBuffer 3 | #*5 μL NEBuffer 3 | ||
#*44-X μL H<sub>2</sub>O | #*44-X μL H<sub>2</sub>O | ||
#Incubate at 55°C for | #Incubate at 55°C for at least 1 hour | ||
#Heat-inactivate at 80°C for 20 mins | #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 MgCl<sub>2</sub> | |||
#*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 MgCl<sub>2</sub> | |||
#*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 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. | |||
|} | |||
Do a purification step? Or use it directly? | |||
__NOTOC__ |
Latest revision as of 10:01, 1 February 2008
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
Quality control procedure
|
Unit test definition
|
Alternative protocol from Shuman et al.From Shuman 1994: maximum topoisomerase activity was observed with a 20 μL reaction mixture containing
|
Do a purification step? Or use it directly?