Plasmid (virus) prep from (50ml culture) Sulfolobus (download as file)
Pellet cells (10 minutes at 5000rpm) and resuspend in 0.8 ml solution 1 (see below)
Add 1.6 ml solution 2, mix by shaking 5 times, put on ice for 5 minutes.
Add 1.2 ml ice-cold solution 3, vortex briefly and leave on ice for 3-5 minutes.
Spin mixture for 30 minutes at 20,000rpm in Sorvall centrifuge (SS-34), place supernatant in a new (phenol resistant) tube.
Add 1 volume (4ml) Phenol, shake 30-40 times, spin 30 minutes at 20,000rpm in SS-34 rotor.
Decant aqueous phase (and a little bit of the interface) and add 1 volume Phenol/Chloroform/Isoamyl-alcohol to it, shake 30-40 times, spin for 20 minutes at 20,000rpm in SS-34 rotor.
Decant aqueous phase and add 1 volume Chloroform to it, shake 30-40 times, spin for 10 minutes at 20,000rpm in SS-34 rotor.
Remove aqueous phase and add 0.7 volumes Isopropanol to it. Leave for 10 minutes at room temperature, then spin for 30 minutes at 20,000rpm in SS-34 rotor.
Wash pellet with 1ml 70% ethanol and allow to air dry.
Dissolve pellet in 50 microliters TE + RNAse (10μg/ml)
Cut 2-5 μl of plasmid with restriction endonuclease
50 mM Glucose
25 mM Tris/HCl pH 8.0 10 mM EDTA
Solution 2 0.2M NaOH 1% (w/v) SDS
60 ml 5M potassium acetate solution 11.5 ml Acetic Acid
28.5 ml ddH2O