Mesoplasma florum:restriction enzyme tests

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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