Difference between revisions of "Mesoplasma florum:restriction enzyme tests"

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(New page: Protocol for quick tests of restriction enzyme activity in novel species * Belavin P. A., Dedkov V.S., Degtyarev S.Kh., A method to detect restriction endonucleases in bacterial colonies. ...)
 
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Lysis buffer
 
Lysis buffer
* 100 mM tris-HCl pH 8.0
+
* 100 mM Tris-HCl pH 8.0
 
* 50 mM NaCl
 
* 50 mM NaCl
 
* 5 mM EDTA
 
* 5 mM EDTA
Line 17: Line 17:
  
  
* Put a colony into 250 ul of lysis buffer
+
* Put a colony or pelleted cells into 250 ul of lysis buffer
* Incubate 15 minutes at room temperature with shaking
+
* Incubate 15 minutes at room temperature with shaking or vortexing
 
* Centrifuge at high speed 1 min
 
* Centrifuge at high speed 1 min
 
* Add 1-4 μL to 20 μL of reaction buffer with 500 ng of lambda DNA
 
* Add 1-4 μL to 20 μL of reaction buffer with 500 ng of lambda DNA
 
* Incubate 1-2 hours at 30°C
 
* Incubate 1-2 hours at 30°C
 
* Run gel
 
* Run gel

Revision as of 11:43, 22 February 2008

Protocol for quick tests of restriction enzyme activity in novel species

  • Belavin P. A., Dedkov V.S., Degtyarev S.Kh., A method to detect restriction endonucleases in bacterial colonies. (1988) Applied Biochemistry and Microbiology (Russia) 24(1):121-124. [1]


Lysis buffer

  • 100 mM Tris-HCl pH 8.0
  • 50 mM NaCl
  • 5 mM EDTA
  • 0.1% Triton X-100


Reaction buffer

  • 20 mM Tris-HCl pH 7.5
  • 50 mM NaCl
  • 10 mM MgCl2
  • 1 mM DTT


  • Put a colony or pelleted cells into 250 ul of lysis buffer
  • Incubate 15 minutes at room temperature with shaking or vortexing
  • Centrifuge at high speed 1 min
  • Add 1-4 μL to 20 μL of reaction buffer with 500 ng of lambda DNA
  • Incubate 1-2 hours at 30°C
  • Run gel