TITLE: Promega Wizard DNA extraction - Drosophila whole body protocol
AUTHORS: finley.thomas.

[DESCRIPTION]

Edited version of the 'Promega Wizard Genomic DNA Purification' protocol optimised to work on pooled whole-body fruit fly samples. 




[STEPS]
SECTION: Lysis &amp; Incubation
1. Add 10 flies into a 1.5ml Eppendorf tube and cover with 300 µL . 

SECTION: Lysis &amp; Incubation
3. Once the flies are throughly homogenised, add another  300 µL . 

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

SECTION: Lysis &amp; Incubation
2. Use a micro-tube pestle to crush the flies until they are fully ground. 
SECTION: Lysis &amp; Incubation
2.1. 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. 
SECTION: Lysis &amp; Incubation
4. Vortex the homogenised solution then incubate at 65 °C . 

SECTION: Lysis &amp; Incubation
5. Remove the samples from the heat and lower the set temperature to 55 °C . 

SECTION: Lysis &amp; Incubation
6. Allow for the samples to cool for a few moments, then add 17.5 µL . 

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

SECTION: Lysis &amp; Incubation
7. Vortex the samples throughly then incubate at 55 °C .  

Vortex the samples regularly throughout this time. 




SECTION: Protein Precipitation
8. Remove the samples from the heat block and allow to cool to room temperature. 
SECTION: Protein Precipitation
9. Add 200 µL to each sample & vortex for 20 seconds (until thoroughly mixed). 

SECTION: Protein Precipitation
10. Leave the samples on ice . 

SECTION: Protein Precipitation
11. Centrifuge the samples at high speed: 13200 rpm . 

SECTION: Protein Precipitation
12. Carefully transfer the supernatant (liquid) to a new (labelled) micro-centrifuge/Eppendorf tube. 

Take care not to disturb the pellet. 
SECTION: Protein Precipitation
13. If any tissue or protein precipitate (white mass) remains in the supernatant, repeat steps 11 & 12. 
SECTION: Protein Precipitation
14. Once the supernatant is clear of tissue mass and protein precipitate, add 600 µL (at room temperature). 

SECTION: DNA elution and cleansing
15. 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. 
SECTION: DNA elution and cleansing
16. Centrifuge the samples at 13200 rpm . 

SECTION: DNA elution and cleansing
17. Taking care not to disturb the pellet (of DNA), remove the supernatant. 

If the supernatant does not contain the pellet, it can be discarded. 
SECTION: DNA elution and cleansing
18. Add 600 µL , then gently invert the tube several times (to wash the DNA). 

SECTION: DNA elution and cleansing
19. Centrifuge at 13200 rpm . 

SECTION: DNA elution and cleansing
20. Making sure that the DNA pellet is not disturbed, remove the ethanol (this can be discarded). 
SECTION: DNA elution and cleansing
21. Invert the (open) tube onto clean absorbent paper; leave for 10-15 minutes. 

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

SECTION: DNA elution and cleansing
22. Add 100 µL  (Nuclease Free Water) and incubate at 65 °C . 

Mix the solution by gently tapping and shaking the tubes. 

SECTION: DNA elution and cleansing
23. Store the DNA at 2-8 °C .