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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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] | ||
| - | + | ==Lysis buffer== | |
| - | Lysis buffer | + | * 100 mM Tris-HCl pH 8.0 |
| - | * 100 mM | + | |
* 50 mM NaCl | * 50 mM NaCl | ||
* 5 mM EDTA | * 5 mM EDTA | ||
* 0.1% Triton X-100 | * 0.1% Triton X-100 | ||
| - | + | ==Reaction buffer== | |
| - | Reaction buffer | + | |
* 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 | ||
| + | * This is essentially NEB Buffer 2, which has pH 7.9 and 10 mM Tris | ||
| - | + | ==Procedure== | |
| - | * 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 | ||
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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
