Plasmid (virus) prep from (50ml culture) Sulfolobus (download as file)

  1. Pellet cells (10 minutes at 5000rpm) and resuspend in 0.8 ml solution 1 (see below)

  2. Add 1.6 ml solution 2, mix by shaking 5 times, put on ice for 5 minutes.

  3. Add 1.2 ml ice-cold solution 3, vortex briefly and leave on ice for 3-5 minutes.

  4. Spin mixture for 30 minutes at 20,000rpm in Sorvall centrifuge (SS-34), place supernatant in a new (phenol resistant) tube.

  5. Add 1 volume (4ml) Phenol, shake 30-40 times, spin 30 minutes at 20,000rpm in SS-34 rotor.

  6. 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.

  7. 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.

  8. 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.

  9. Wash pellet with 1ml 70% ethanol and allow to air dry.

  10. Dissolve pellet in 50 microliters TE + RNAse (10μg/ml)

  11. Cut 2-5 μl of plasmid with restriction endonuclease

Solution 1
50 mM Glucose
25 mM Tris/HCl pH 8.0 10 mM EDTA

Solution 2 0.2M NaOH 1% (w/v) SDS

Solution 3
60 ml 5M potassium acetate solution 11.5 ml Acetic Acid
28.5 ml ddH2O