Plasmid Rescue of PZ Element
1. Add 60 flies to a 2059 tube containing 1.5 mL of homogenization buffer: 0.1 M Tris, pH 9, 0.1 M EDTA, 1% SDS. Homogenize with the Polytron (small tip--autoclave before use) until flies are finely ground and solution is strongly colored.
2. Centrifuge at 3500 rpm for 5 minutes. Transfer 1 mL of supernatant into an Eppendorf, avoiding the cloudy material at the top of the liquid.
3. Add 140
l of 8 M KAc and chill on ice for 30 m.4. Spin @ 4°C for 10 m. and transfer as much supernatant as possible to a new tube.
5. Extract with 600
l of phenol/CHCl3.6. Add 0.5 volumes (400-450
l) of isopropanol and sit at RT for 5 m.7. Spin at RT for 10 m. and carefully aspirate supernatant.
8. Wash with 1 mL of 70% EtOH, dry, and resuspend pellet in 70
l of TE+RNase A. This gives approximately 0.5 fly equivalents per l.9. Digest 4
l of the DNA thus produced (~2 flies worth) with XbaI alone, XbaI+NheI, or XbaI+SpeI in a 50 l reaction.10. Add 100
l of phenol/CHCl3 to each digest, then 50 l of H2O. Vortex, spin, transfer the aqueous phase.11. Add 1
l of 20 mg/mL glycogen and 10 l of 3 M NaAc to each digest, then 200 l of 100% EtOH. Incubate @ -20°C, 30 m.12. Spin @ 4°C, 10 m. Aspirate & wash with 70% EtOH, dry, and resuspend in 20-50
l of TE.13. Add 80
l of 5X T4 DNA ligase buffer (+ATP, if necessary, to 0.9 mM), raise volume to 399 l with H2O, and add 1 l of T4 DNA ligase. Incubate O/N at 16-18°C.14. Extract ligations with phenol/CHCl3, transferring the aqueous phase.
15. Add 100
l of 10 M NH4Ac, mix, and add 1 mL of 100% EtOH. Incubate @ -20°C, 30 m.16. Spin @ 4°C, 10 m. Aspirate & wash with 70% EtOH, dry, and resuspend sterilely in 5
l sterile ddH2O.17. Transform into E. coli by one of the high-efficiency methods-- CaCl2 transformation of Stratagene supercompetent bugs has so far been most successful for me--and plate on LB plates with 50
mg/mL kanamycin.