Knight:TOPO vector preparation: Difference between revisions

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===Covalently attach DNA topoisomerase I from ''Vaccinia''===
===Covalently attach DNA topoisomerase I from ''Vaccinia''===
#Mix
{|
|
====Quality control procedure====
#Mix a 50 μL reaction including
#*50 mM Tris-acetate (pH7.5)
#*50 mM Tris-acetate (pH7.5)
#*100mM NaCl
#*100 mM NaCl
#*2.5 mM MgCl<sub>2</sub>
#*2.5 mM MgCl<sub>2</sub>
#*0.1 mM EDTA
#*0.1 mM EDTA
#*1 &mu;L DNA topoisomerase I from ''Vaccinia''
#*1 &mu;L DNA topoisomerase I from ''Vaccinia''
#Incubate at 37&deg;C for at least 1 hour
#Incubate at 37&deg;C for at least 1 hour
 
|
Do a purification step? Or use it directly?
====Unit test definition====
 
#Mix
===ATP?===
#*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&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  
From Shuman 1994: maximum topoisomerase activity was observed with a 20 &mu;L reaction mixture containing  
*50 mM Tris-HCl (pH 8.0)
*50 mM Tris-HCl (pH 8.0)
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*0.3 &mu;g of plasmid DNA
*0.3 &mu;g of plasmid DNA
*0.63ng of ''Vaccinia'' topoisomerase I, incubated at 37&deg;C for five minutes.
*0.63ng of ''Vaccinia'' topoisomerase I, incubated at 37&deg;C for five minutes.
|}
Do a purification step? Or use it directly?


On 7/19, I tried the above mixture, except I used 5 &mu;L DNA and 1 &mu;L enzyme. Also, the incubation time was for fifteen minutes instead of five. [[User:Yeem|yeem]] 15:31, 19 July 2007 (EDT)
__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

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

  1. 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
  2. Incubate at 37°C for at least 1 hour

Unit test definition

  1. 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
  2. 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?


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