This small scale preparation will yield approximately 5 ug of genomic DNA. As long as the specific activity of the probe is around 3 X 10⁸ cpm/ug, 0.5 ug of this genomic DNA should be sufficient on a Southern Blot to detect a single copy gene with an overnight exposure.  I've made a significant modification to the procedure at step 3.  I recommend homogenizing the flies by hand rather than with the motorized pestle.  This provides more consistent preparation of high molecular weight DNA.

Have available a solution of Fly Homogenization Buffer (stored at room temperature) and a solution of cold 8 M potassium acetate.

Recipe for 100 ml of Fly Homogenization Buffer [0.2 M Sucrose, 0.1 M Tris base, 50 mM Na2EDTA, 0.5% SDS: adjust the pH of the final mixture to 9.2]:

6.85 g sucrose

1.21 g Tris base

1.86 g Na2EDTA

0.5 g SDS

Adjust pH to 9.2 with 10 M NaOH.

1. Put 30 anesthetized flies into a 1.5 ml microfuge tube. These can be processed immediately or stored in the freezer for up to a few weeks.
2. Add 250 ul of homogenization buffer.
3. Hand grind the flies with a small blue plastic pestle.  Move the pestle up and down and twirl it.  Spend no more than 2 minutes per samples.  We used to use a motorized pestle, but I think this was shearing the DNA.  The pestles are washed thoroughly with soap and water. If you are having trouble with contamination (e.g. PCR reactions) you might consider soaking the pestles for 20 minutes in 1M HCl and then for 20 minutes in 1M NaOH. This depurinates and cleaves DNA.
4. Add 250 ul homogenization buffer to the flies, cap and invert tube several times to mix.
5. Heat homogenate at 70^oC for at least 10'. During this time, thoroughly clean the pestles with detergent and then water.
6. Add 75 ul of cold 8 M potassium acetate. Mix and incubate on ice for 10'. A brown precipitate often settles to the bottom of the 8 M potassium acetate stock solution. This impurity is from the solid potassium acetate and should not be disturbed. The potassium acetate solution could be filtered but this is not necessary.
7. Microfuge sample for 10' at 4^oC. With a blue pipette, transfer 400 ul of the supernatant to a new tube. A very large pellet should remain and be discarded.
8. Add 1 ml of ethanol to the solution and mix by inverting the tube. Incubate at room temperature for 2' and then microfuge at room temperature for 5'. A small precipitate should be evident.
9. Discard supernatant and disperse the pellet in 200 ul of 100 mM NaCl, 10 mm Tris-Cl pH 8, 1 mM EDTA (this can be approximated by combining 9 parts TE with 1 part 1M NaCl).  Then add 2 ul of 10% SDS and 10 ug of RNase A.  Mix throughly and incubate at 37^oC for at least 1 hour, triterating intermittently if possible.  After 1 hour, add 10 ug of proteinase K and incubate at 37^oC for at least 1 hour.  The sample can be incubated overnight.  Note that I have made several changes at this step.  NaCl and SDS have been added to help dissolve the nucleic acid precipitate.  Proteinase K digestion has been added to simplify the subsequent phenol extractions.  The original procedure called for dissolving the nucleic acid in TE, RNAse digesting and then proceding onto the phenol extractions.
10. Extract 2 times with equilibrated phenol. For each extraction, add 200 ul of equilibrated phenol and shake vigorously for at least 30 seconds.  Separate the phases by microfuging at room temperature for 5'. After the first extraction, transfer 180 ul of the aqueous phase (top phase) to a fresh tube. After the second extraction transfer 160 ul of the aqueous phase to another fresh tube. Try to avoid the cloudy material that collects at the interface after each extraction.
11. Extract 1 time with 200 ul of ether. Shake 30 seconds, centrifuge 2' and discard top phase (ether) into a beaker in the hood.
12. Leave the tubes uncapped and float them in hot tap water for few minutes to evaporate residual ether.  You can determine that sufficient ether has evaporated when the aqueous layer "sticks" to the sides of the eppendorf tube if tilted.
13. Add 16 ul of 3M sodium acetate (pH 6 to 7) and mix. Then add 400 ul of cold 100% ethanol (stored at -20^oC). The DNA will begin to precipitate at the interface between the ethanol and the aqueous layer. Slowly invert the tube several times to achieve mixing. A stringy clump of DNA should form. If no precipitate is observed, the preparation probably failed.
14. If possible, try to remove the liquid with a pipette and leave the clump of DNA behind.  Alternatively, the samples can be centrifuged briefly to pellet the DNA.  Add 200 ul of cold 75% ethanol to the clump of DNA and invert the tube so that the inside surface of the tube is rinsed with ethanol. Try to keep the clump of DNA at the bottom of the tube.  With the tube upright, carefully remove the ethanol wash solution. Microfuge the sample briefly and remove the residual wash solution that has accumulated at the bottom of the tube.  Set the tube on its side and allow the DNA pellet to air dry.
15. Dissolve the DNA in 50 ul of 10 mM Tris-Cl pH 8, 1 mM EDTA. The yield is usually around 5 ug of genomic DNA from 30 flies. Quantify the DNA using the fluorometer. It should be ready for PCR or restriction digestion.