Mesoplasma florum:restriction enzyme tests

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(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. ...)
Line 4: Line 4:
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 15:43, 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
Personal tools