Virginie Orgogozo

Preparation of genomic DNA
(BDGP protocol, described in the blue book “Drosophila protocols”)

good to amplify 2kb-PCR fragments, the result is a mix of genomic DNA+RNA (no RNAse step)

buffer A:
        100mM Tris-Cl pH=7.5
        100mM EDTA
        100mM NaCl
        0.5% SDS
(stored at room temperature)

buffer B:
        200mL of 5M potassium acetate
        5000mL of 6M lithium chloride
mix together before use, store at 4C.

1. collect 30 flies in a 1.5mL microcentrifuge tube and freeze the tube for 5min.
2. grind flies in 200uL of buffer A with a disposable tissue grinder. Add an additional 200uL of buffer A and continue grinding until only cuticles remain (1-2min by hand).
3. incubate at 65C for 30min.
4. add 800uL of buffer B, mix well by inverting the tube multiple times, then incubate on ice for at least 10min and up to a few hours.
5. centrifuge at 12 000rpm for 15min.
6. transfer the supernatant to a new tube and centrifuge again at 12 000rpm for 15min. Transfer the supernatant to a new tube (make sure you don’t put white tissues).
7. add 600uL of isopropanol and vortex.
8. centrifuge at 12 000rpm for 15min.
9. remove supernatant and wait ON until the pellet dries. (Otherwise wash pellet in 20% EtOH).
10. resuspend in 150uL of milliQ water.
Usually the final DNA concentration is around 25ug/uL.