Isolating Genomic DNA from Drosophila
Based on Berkeley fly project protocol
 Start waterbath – turn to 65º C
 Knock out 30 flies and put into 1.5 ml microcentrifuge tube.
 Add 200 µL Buffer A [100 mM Tris-HCl pH 7.5, 100 mM EDTA, 100 mM
NaCl, 0.5 % SDS).
 Grind with Kontes “Pellet Pestle.”
 Add another 200 µL Buffer A and grind again. There will be lots of cuticle
parts, but fly bodies should no longer be recognizable.
 Put in 65º C water bath for 30 min. and make KOAc/LiCl solution.
 Put at room temp, add 800 µL KOAc/LiCl solution [1.43 M KOAc, 4.28 M
LiCl]
 Cap, mix well, put on ice for 10 min for protein and detergent to precipitate.
 Spin in microfuge for 15 min at room temp. Carefully transfer the
supernatant to a new tube; some floating junk may come along.
 Spin again for 15 minutes to pellet any junk that transferred.
 Very carefully remove 1 mL of the supernatant to a new tube. Add 600 µL
of isopropanol. Cap, mix well.
o If it’s easier, remove 900 µL of supernatant and add 540 µL
isopropanol.
 Spin in microfuge for 10 minutes and then remove the supernatant.
 Wash the pellet with 1 mL of 70% EtOH. Spin in microfuge for a few
seconds, then remove the last traces of EtOH by tapping on kimwipe.
o Alternatively, tubes can be spun in microfuge for 3 minutes after
adding ethanol. Remove the last traces of EtOH as above.
 Add 150 µL TE to redissolve the DNA. Put at 65º C for 5 min. to help it
dissolve. When dissolved, freeze to store it.
Making buffer A (10 mL):
1 mL 1M Tris-HCl pH 7.5
2 mL 0.5M EDTA
200 µL 5M NaCl
500 µL 10% SDS
6.3 mL distilled water
Making KOAc/LiCl solution (8.75 mL):
2.5 mL 5M KOAc
6.25 mL 6M LiCl