Apr 24, 2023
Promega Wizard DNA extraction - Drosophila whole body protocol
- 1UCL
Protocol Citation: Finley Grover-Thomas 2023. Promega Wizard DNA extraction - Drosophila whole body protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjrzdlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 21, 2023
Last Modified: April 24, 2023
Protocol Integer ID: 80886
Keywords: Promega, DNA extraction, Wizard, Drosophila, Insect
Abstract
Edited version of the 'Promega Wizard Genomic DNA Purification' protocol optimised to work on pooled whole-body fruit fly samples.
Lysis & Incubation
Lysis & Incubation
Add 10 flies into a 1.5ml Eppendorf tube and cover with 300 µL Nuclei Lysis Solution .
2m
Use a micro-tube pestle to crush the flies until they are fully ground.
5m
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 300 µL of Nuclei Lysis Solution .
By this point, the solution should be opaque and red-brown in colour.
2m
Vortex the homogenised solution then incubate at 65 °C for 20 minutes .
20m
Remove the samples from the heat and lower the set temperature to 55 °C .
Allow for the samples to cool for a few moments, then add 17.5 µL Proteinase K .
(This is not included in the Promega Wizard kit).
2m
Vortex the samples throughly then incubate at 55 °C for 3 hours .
Vortex the samples regularly throughout this time.
3h
Protein Precipitation
Protein Precipitation
4m
4m
Remove the samples from the heat block and allow to cool to room temperature.
Add 200 µL Protein Precipitation Solution to each sample & vortex for 20 seconds (until thoroughly mixed).
Leave the samples On ice for 5 minutes .
5m
Centrifuge the samples at high speed: 13200 rpm , for 4 minutes .
4m
Carefully transfer the supernatant (liquid) to a new (labelled) micro-centrifuge/Eppendorf tube.
Take care not to disturb the pellet.
2m
If any tissue or protein precipitate (white mass) remains in the supernatant, repeat steps 11 & 12.
Once the supernatant is clear of tissue mass and protein precipitate, add 600 µL isopropanol (at room temperature).
2m
DNA elution and cleansing
DNA elution and cleansing
31m
31m
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.
5m
Centrifuge the samples at 13200 rpm , for 1 minute .
1m
Taking care not to disturb the pellet (of DNA), remove the supernatant.
If the supernatant does not contain the pellet, it can be discarded.
4m
Add 600 µL 70% ethanol , then gently invert the tube several times (to wash the DNA).
3m
Centrifuge at 13200 rpm , for 1 minute .
1m
Making sure that the DNA pellet is not disturbed, remove the ethanol (this can be discarded).
2m
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 .
15m
Add 100 µL DNA Rehydration Solution (Nuclease Free Water) and incubate at 65 °C for 1 hour .
Mix the solution by gently tapping and shaking the tubes.
Store the DNA at 2-8 °C .