In a culture tube, inoculate 5ml of LB broth (with correspondent antibiotic) with a colony and incubate overnight at 37C with gentle shaking (about 60rpm).
Transfer 1.5ml of the overnight culture in a 1.5ml Eppendorf tube and centrifuge for 2 min at 5000rpm. (repeat once more with another 1.5 ml in the same tube). If desired, keep the remaining for a glycerol stock (see below)
Discard the supernatant, and let the tube upside-down on a clean tissue (tela or similar) to completely remove the media
Add 200μl of the cell Resuspension Solution and resuspend thoroughly by vortexing. IMPORTANT: from this point on, DON'T vortex or vigorously shake the samples!
Add 200μl of the Cell Lysis Solution and immediately mix by inverting the tubes 4-5 times. Incubate until solution clears (approx. 1-5 min)
Add 10μl of Alkaline Protease Solution and mix by inverting the tube 4-5 times. Incubate at room temperature for 5min (Do not incubate longer!)
Add 280μl of the Neutralization Solution, immediately mix by inverting the tube 4-5 times Centrifuge for 5-10min at max speed
Transfer the supernatant in a new 1.5ml Eppendorf tube and add 100% ethanol at -20°C until filled Incubate on ice for exactly 2min
Centrifuge for 30min at 4C at max speed
Discard the supernatant and wash the pellet with 500μl of 70% ethanol
Air dry the pellet and resuspend in 100μl of TE (not for sequencing!!) or water.
(optional) Add RNAsa to a final concentration of 10μg/ml (2μl enzyme (500μg/ml) in 100 μl buffer) and incubate at room temperature for 3 hours.
Add 300 μl Glycerol 50% to a 1.5 ml tube
Add 700 μl of the cell culture
Mix and freeze in liquid nitrogen
Store at -80°C