Hermann Steller

RAPID SMALL SCALE ISOLATION OF DROSOPHILA DNA AND RNA


DNA:
isolation of Drosophila DNA; DNA isolation;
The following protocol describes the isolation of DNA from adult flies; but can be used equally well to extract DNA from other developmental stages. DNA prepared according to that method is readily digested by restriction enzymes and has an average size of 40-60 kb. In order to remove RNA contaminating the DNA preparations DNase free RNase should be included when digesting with restriction enzymes.

1) Anesthesize flies with CO2 or ether and put 1-20 flies in an eppendorf tube. Keep on ice until next step.

2) Add solution A containing 0.1 M Tris-HCl, pH 9.0; 0.1 M EDTA; 1% SDS and 0.5-1% DEPC (added directly before use) and homogenize gently with a 3 mm diameter glass or metal rod . Use 100 µl of solution A for extracting DNA from 1-5 flies, 200 µl for 6-10 flies and 500 µl for up to 50 flies. Incubate for 20-30 minutes at 70°C.

3) Add 14 µl of 8 M potassium acetate for each 100 µl homogenate and leave on ice for 30 minutes.

4) Spin in the eppendorf centrifuge at 4°C for 15 minutes. Transfer the supernatant into a fresh eppendorf tube being careful not to disturb the pellet. If you get flakes in the supernatant respin.

5) Precipitate DNA by adding 0.5 volumes of isopropanol at room temperature and spin for 5 minutes at RT. Wash the pellet carefully with 70% EtOH, respin, dry and redissolve in 10 (1 fly) to 100 (50 flies) µl TE.


RNA:
RNA isolation; isolation of Drosophila RNA;
This is a modification of the method of Chirgwin et al. (Biochemistry 18, 5294-5299, 1979) which can be used for the rapid extraction of total RNA form all developmental stages of Drosophila.

1) Homogenize tissue (e.g. 1 fly or 50-100 embryos in 50 µl and up to 200 flies in 1 ml) GHCl buffer (7.5 M guanidium hydrochloride; 0.025 M NaOAc, pH 7.0; 5 mM DTT) + 0.5% N-laurylsarcosinate + 0.5% DEPC.

2) Extract once with an equal volume of phenol-chlorororm and separate phases by centrifugation.

3) Transfer the aqueous (upper) phase into a new tube and precipitate the RNA by adding 1 µl 1 M acetic acid and 25 µl ethanol for each 50 µl of GHCl-solution. Leave at -20°C for 3-24 hours. Pellet the RNA by centrifugation for 5 min., 8-12K.

4) Remove supernatant as complete as possible and redissolve the pellet in half of the original volume (but minimally 50 µl) GHCl buffer.

5) Reprecipitate the RNA as in step 3. (The reprecipitation serves to remove DNA which will not precipitate under these conditions).

5a) optional: reprecipitate the RNA for 1-2 more times by repeating steps no. 3 and 4. This is only necessary if large amounts of DNA have to be removed, e.g. when analysing the transient expression of DNA injected into embryos.

6) Wash (and store) the RNA pellet in 100% ethanol, room temperature. For Northern analysis the RNA is best dissolved directly in Northern sample buffer (e.g. 50% formamide, 2.2 M formaldehyde, 1x MOPS).