2) Set a porcelain mortar in dry ice, pour in liquid Nitrogen, and immerse the pestle until cold.
3) Add frozen flies, grind as a slurry in liquid Nitrogen, then allow the Nitrogen to boil off.
4) Chill a 50 ml polypropylene Oak Ridge tube; and a spatula in liquid Nitrogen, then add up to 5 ml of the frozen fly powder to the cold tube on dry ice.
5) Remove the tube from the dry ice and immediately add 15 ml Homogenization Buffer (HB) and 15 ml 1:1 Phenol/Chloroform.
6) Cap the tube and mix by gentle inversion (to minimize DNA shearing;) until well
mixed and then for 30 minutes on a lab-quake or similar.
WARNING - EXPLOSION HAZARD
AS THE FROZEN POWDER DISPERSES IT WILL RELEASE NITROGEN GAS. YOU
MUST PERIODICALLY CRACK THE CAP TO RELEASE THE PRESSURE AS THE
POWDER IS DISPERSED, UNTIL IT IS ALL DISSOLVED. OTHERWISE THE TUBE
MAY EXPLODE AND SPRAY NOXIOUS CHEMICALS ON YOU.
7) Spin at 18,000 rpm at 20°C for 10 minutes (if at 4°C the Urea and SDS in the HB will precipitate).
8) Take the aqueous (top) phase, avoiding the interface, to a new tube and repeat the extraction twice more.
9) Take the aqueous (top) phase, avoiding the interface, to a new tube and add two
volumes of ethanol (don't add salt, it's already in the HB), mix and spin at 18,000
rpm at 20°C for 10 minutes (if at 4°C the Urea and SDS in the HB will precipitate).
10) Resuspend the pellet in 3 ml TE by gentle inversion on a lab-quake or similar (this
will take some time).
11) Add 3g CsCl and 0.3 ml 10mg/ml ethidium bromide, fill a 5ml ultracentrifuge tube (top up with the same solution less the DNA pellet), check the density (³ = 1.56) and spin at 45,000 rpm, 15°C for 16 hours.
12) Remove the DNA band by side-puncture (avoiding the pelleted RNA) and remove the ethidium by repeated extraction with CsCl saturated butan-2-ol.
13) Dilute 3 fold with TE, add 1/10 volume 5M NaCl and precipitate with 2 volumes of ethanol.
14) Wash the pellet in 70% ethanol, and resuspend in TE by gentle inversion.
15) Quantify by UV absorbance.
HB Buffer;:
7 M Urea
2 % SDS
50 mM Tris pH 7.5
10 mM EDTA
0.35 M NaCl
Notes-
This protocol works by keeping the material frozen until it hits the combined HB
and phenol/chloroform. Thus the cells "wake-up" into a chemical environment that
immediately denatures all membranes and proteins. In other words, the nucleases never
get a chance. This method works well as an RNA prep ;also, just vortex at each mixing
step, extract more exhaustively (until the interface is totally clean) and don't Caesium
band. The resulting total nucleic acids prep can be used to make polyA+ RNA; on an
oligodT column, for Northerns;, but if it is important to remove the DNA completeley (ie
for cDNA cloning;) then some measure must be taken to achieve this. This prep can be
scaled down to 20 flies or fewer in a microfuge tube. For the mini-prep; don't freeze, just
homogenize in 0.5 ml HB and then extract, twice with phenol/chloroform, ethanol
precipitate twice, wash in 70% ethanol and resuspend in TE. When you digest the
mini-prep DNA with restriction enzymes add boiled RNAse; to 0.1 mg/ml.