Caution! In converting these protocols to HTML, some units have been scrambled.
1. Grind 20-50 frozen flies with a mortar and pestle in N2(l) and suspend in 2 ml Lysis Buffer.
2.Add Proteinase K to 100 mg/ml; incubate 1-2h @ 37oC with occassional swirling.
3. Phenol extract GENTLY, 10'; cfg. 5' @ RT. Harvest the aqueous phase with a large bore transfer pipet.
4. Phenol-Sevag extract 1X; Sevag extract 1X.
5. Harvest the aqueous phase and precipitate the DNA with 0.5 vol 7.5M NH4OAc + an equal vol isopropanol; mix gently.
6. Spool out the DNA (or leave to ppt. ON @ -20oC), rinse in EtOH and air dry briefly before resuspending in TE.
7. Digest the DNA with pretreated RNase A @ 100mg/ml; 37oC, 30'.
8. Phenol-Sevag extract 1X; Sevag extract 1X.
9. NaOAc/EtOH ppt.
10. Resuspend GENTLY in TE-4.
Lysis Buffer 50 mls:
100 mM Tris-HCl, pH 8 5.0 ml 1M Tris-HCl
50 mM NaCl 0.5 ml 5M NaCl
50 mM EDTA 5.0 ml 0.5M EDTA
1 % SDS 2.5 ml 20% SDS
150 mM Spermine 75 ml 100mM Spermine-HCl4
500 mM Spermidine 50 ml 500mM Spermidine
36.875 ml ddH2O
TE (10 mM Tris-HCl, pH 8/1 mM EDTA)
TE-4 (10 mM Tris-HCl, pH 8/0.1 mM EDTA)