Mesoplasma florum:restriction enzyme tests: Difference between revisions
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* 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 | |||
* Put a colony or pelleted cells into 250 ul of lysis buffer | * Put a colony or pelleted cells into 250 ul of lysis buffer | ||
Revision as of 12:11, 25 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
- This is essentially NEB Buffer 2, which has pH 7.9 and 10 mM Tris
- 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
