Mesoplasma florum:restriction enzyme tests-PDF
Material
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 for 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