The plasmid to be used has to have f1 origin of replication and the bacterial strain has to have an F' episome.

1. Take a single colony and put in 5 ml of 2 x TY + antibiotic + 30 g/ml Chloramphenicol (if using CJ236). Add 5 l of helper phage, VCSM13 (Stratagene), ~1 x 1011 pfu/ml.

2. Incubate 1h 20 min @ 37oC with shaking. Add kanamycin to 70 g/ml (to maintain the phage). Continue to incubate for 12-13 h.

3. Collect 4 x 1 ml, 5 min, Eppendorf centrifuge.

4. To each 1 ml of supernatant add 150 l 20 %PEG6000/2.5 M NaCl. Incubate 20 min on ice.

5. Centrifuge 12000 rpm, Eppendorf, 5 min. Remove supernatant; centrifuge again and remove the residual super carefully.

6. Resuspend in 2 x 400 l 0.3 M NaAc, pH5.2/1 mM EDTA.

7. Add x 400 l Phenol/Chloroform. Extract.

8. Add 1 ml of EtOH to the aqueous phase. Incubate 5 min RT.

9. Centrifuge 10 min 12000 rpm. Dry pellet and dissolve in 40 l TE. Yield should be ~ 100 ng/l. Small amount of phage DNA (6 kb if VCSM13) might be isolated together with the ss plasmid.