Welcome to the Home of Vesicle Trafficking
A small repository of synaptic protein information
Caution! In converting these protocols to HTML, some units have been
scrambled.
Specifically, microliters (ul) often appear as milliliters (ml).
Caution! In converting these protocols to HTML, some units have been
scrambled.
Specifically, microliters (ul) often appear as milliliters (ml).
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)
acc 1/90
