Promega Wizard DNA extraction - Drosophila whole body protocol

Add 10 flies into a 1.5ml Eppendorf tube and cover with 300µL .

Use a micro-tube pestle to crush the flies until they are fully ground.

For the initial crushing, use your hands and the pestle only.

Once the flies are partially disintegrated an electronic homogeniser can be used to increase efficiency.

Take care not to froth the solution up too much.

Once the flies are throughly homogenised, add another 300µL .

By this point, the solution should be opaque and red-brown in colour.

Vortex the homogenised solution then incubate at 65°C .

Remove the samples from the heat and lower the set temperature to 55°C .

Allow for the samples to coo l for a few moments, then add 17.5µL .

(This is not included in the Promega Wizard kit).

Vortex the samples throughly then incubate at 55°C .

Vortex the samples regularly throughout this time.

Remove the samples from the heat block and allow to cool to room temperature.

Add 200µL to each sample & vortex for 20 seconds (until thoroughly mixed).

Leave the samples On ice .

Centrifuge the samples at high speed: 13200rpm,0h 0m 0s .

Carefully transfer the supernatant (liquid) to a new (labelled) micro-centrifuge/Eppendorf tube.

Take care not to disturb the pellet.

If any tissue or protein precipitate (white mass) remains in the supernatant, repeat steps 11 & 12. repeat steps 11 & 12.

Once the supernatant is clear of tissue mass and protein precipitate, add 600µL (at room temperature).

Gently invert the samples to mix the isopropanol and supernatant.

White threads of DNA may or may-not form; if no threads are visible after ~5 minutes, continue on with the protocol.

Centrifuge the samples at 13200rpm,0h 0m 0s .

Taking care not to disturb the pellet (of DNA), remove the supernatant .

If the supernatant does not contain the pellet, it can be discarded.

Add 600µL , then gently invert the tube several times (to wash the DNA).

Centrifuge at 13200rpm,0h 0m 0s .

Making sure that the DNA pellet is not disturbed, remove the ethanol (this can be discarded).

Invert the (open) tube onto clean absorbent paper; leave for 10-15 minutes .

T owards the end of this time, set the heating bloc to 65°C .

Add 100µL (Nuclease Free Water) and incubate at 65°C .

Mix the solution by gently tapping and shaking the tubes.

Store the DNA at 2-8°C .