Mesoplasma florum:restriction enzyme tests: Difference between revisions
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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 | * 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 12:43, 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
