Mesoplasma florum:restriction enzyme tests

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.

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