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. ...)
Current revision (12:19, 23 February 2009) (view source)
 
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{{back to protocols}}
Protocol for quick tests of restriction enzyme activity in novel species
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. [http://science.sibenzyme.com/article12_article_14_1.phtml]
* 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. [http://science.sibenzyme.com/article12_article_14_1.phtml]
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==Lysis buffer==
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Lysis buffer
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* 100 mM Tris-HCl pH 8.0
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* 100 mM tris-HCl pH 8.0
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* 50 mM NaCl
* 50 mM NaCl
* 5 mM EDTA
* 5 mM EDTA
* 0.1% Triton X-100
* 0.1% Triton X-100
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==Reaction buffer==
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Reaction buffer
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* 20 mM Tris-HCl pH 7.5
* 20 mM Tris-HCl pH 7.5
* 50 mM NaCl
* 50 mM NaCl
* 10 mM MgCl<sub>2</sub>
* 10 mM MgCl<sub>2</sub>
* 1 mM DTT
* 1 mM DTT
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* This is essentially NEB Buffer 2, which has pH 7.9 and 10 mM Tris
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==Procedure==
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* Put a colony into 250 ul of lysis buffer
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* Put a colony or pelleted cells into 250 ul of lysis buffer
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* Incubate 15 minutes at room temperature with shaking
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* 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
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[[category:Mesoplasma florum]][[category:protocol]]

Current revision

back to protocols

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
  • This is essentially NEB Buffer 2, which has pH 7.9 and 10 mM Tris

Procedure

  • 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
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