Axenic flies

Drosophila genomic DNA extraction

1) Collect 20-50 flies in a tube. Can keep collected flies at -20 if necessary.

2) Homogenize in 350ul of Buffer A. If 40-50 flies, spin down 1000rpm 1 minute and take super.

3) Add 350ul of Buffer 2, mix.

4) Add RNAse A to 20ug/ml. Incubate 30 min at 37C.

5) Add proteinase K to 100ug/ml. Incubate 1 hour 56C (can do 37C but 56C is preferable).

6) Phenol extraction: add 600ul of Phenol:chloroform:isoamyl alcohol (25:24:1) mixture, incubate 20 min with rotation.

Spin down 10000g, take upper phase.

7) Repeat step 6.

8) Add 700ul of isopropanol to the upper phase after second phenol extraction. Mix and incubate 5 min, spin down 5 min 10000g.

9) Remove super( DNA is in the pellet).

10) Wash pellet twice with 70% ethanol (500ul).

11) Wash pellet once with 96% ethanol (500ul).

12) Let the pellet dry for about 10 minutes, and dissolve in 30-40ul of TE or water. 

13) Store at 4C.

Buffer A:

Tris-HCl, pH 7.5   10mM

NaCl                      60mM

EDTA                     10mM

Spermidine            0.15mM

Sucrose                  5%

Buffer B:

Tris-HCl, pH 9.0   200mM

EDTA                     30mM

SDS                       2%

Sucrose                  5%

Phenol-chloroform-isoamyl alcohol:

Prepare phenol-chloroform-isoamyl alcohol (25:24:1) 

May be stored under 100 mM Tris Cl (pH 8.0)