The protocol is based on the permanganate procedure.  When we were using rosy-minus derived lines, we used to use DNA isolated from soft body tissues. Unfortunately, the level of background cutting in tissue from yellow, white derivatives seems high so we have resorted to preparing the DNA for the naked DNA controls from salivary glands.

1. Place 300 ul of dissection buffer into a siliconized 1.7 ml tube.  Place the tube on ice.
2. Dissect salivary glands from 3rd instar larvae and transfer the glands to the siliconized tube.
3. After about 30 to 60 minutes of dissection (or when you have 10 to 20 pairs of glands), add 100 ul of 2X MnO₄ stop mix.
4. Mix thoroughly at room temperature for about 5 minutes.
5. Add 20 ug of proteinase K and digest at 45 to 55^oC for 2 hours.
6. Extract with 400 ul of Phenol, then 400 ul of phenol/chloroform, and finally 400 ul of chloroform.  Remove the organic phase by pipetting it out from underneath the aqueous phase.  If there is a lot of debris at the interface, transfer the aqueous phase to a fresh tube.
7. Add 40 ul of 3M NaOAc pH 7 and 1 ml of ethanol.  Mix thoroughly and place on ice for at least 15 minutes.
8. Microfuge in the cold for 20 minutes - a pellet consisting mostly of RNA should be visible.
9. Discard the supernatant and rinse the precipitate with 75% ethanol.  Spin again and discard supernatant.
10. Air dry the precipitate.
11. Dissolve the precipitate in 20 ul of TE and quantify the DNA with the fluorometer (contaminating RNA does not interfere with the fluorometer measurements).

Dissection buffer (refrigerate): 130 mM NaCl, 5 mM KCl, 1.5 mM CaCl₂

1ml of 2X MnO₄ stop mix:

• 40 ul 1 M Tris pH 7.5
• 8 ul 5M NaCl
• 160 ul 0.5 M NaEDTA
• 200 ul 10% SDS
• 56 ul beta-mercaptoethanol
• 536 ul of H₂O