Oct 31, 2023

Public workspaceDNA extraction - Zooplankton - 96 wells V.2

DOI
dx.doi.org/10.17504/protocols.io.j8nlk4x7wg5r/v2
  • coline.royaux1,2,3,
  • Nicolas Rabet1,2,3,
  • Céline Bonillo1,2,3
  • 1Sorbonne Université;
  • 2Muséum National d'Histoire Naturelle;
  • 3UMR BOREA
Coline Royaux
Open access
DOI: dx.doi.org/10.17504/protocols.io.j8nlk4x7wg5r/v2
Protocol Citation: coline.royaux, Nicolas Rabet, Céline Bonillo 2023. DNA extraction - Zooplankton - 96 wells. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk4x7wg5r/v2Version created by Coline Royaux
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: October 31, 2023
Last Modified: October 31, 2023
Protocol Integer ID: 90207
Keywords: DNA, Extraction, crustacean, freshwater, PNDB, BOREA
Abstract
This protocol was used to extract DNA from whole or parts of zooplanktonic freshwater crustaceans (Copepoda, Branchiopoda, ...) from New Caledonia.
Prepare your 96-well extraction plate with one individual per well. Alternate genus in the wells to detect eventual contamination between wells.
1d
Collect one individual from a sample
1m
Note its genus and determine its sex with a binocular microscope
1m
For big individuals (more than 5 mm), dissect a few legs and put it in the well. Be careful not to damage the rest of the body and put it in a tagged Eppendorf tube.
For little individuals (less than 5 mm), put the whole body.
Note
If necessary, use alcool to get the biological material to fall at the bottom of the well

3m
When all 96 wells are filled, the biological material has to dry to go to lysis

Note
If necessary, use a micropipette to empty an excess of alcool in the well

Safety information
Make sure the plate is closed when you want to transport it elsewhere



12h
Prepare the lysis
15m
Mix Amount18 mL T1 buffer and Amount2.5 mL K proteinase in a Multi-Channel Reservoir and distribute Amount200 µL of the mix with a multimicropipette in each well
Note
Amount180 µL T1 buffer and Amount25 µL K proteinase in each well


10m
Close your extraction plate with a heated aluminium foil and an adhesive plastic film
3m
Put your plate in a proofer at Temperature56 °C DurationOvernight (6h or more) to lyse the tissues
6h
Perform the DNA extraction with a DNA extraction robot
Remove the adhesive film and aluminium foil from the plate and put it in the robot
It will deposit Amount200 µL BQ1 buffer and Amount200 µL ethanol in each wells and mix it

Then, Amount600 µL of the wells content (lysate, BQ1, ethanol) are transfered on the tissue binding plate. Reagents excess are emptied in a waste container.

The tissue binding plate is then dried by a Duration00:05:00 aspiration to bind DNA to the silica membrane of the binding plate
5m
The silica membrane is then washed with Amount600 µL BW buffer and twice with Amount900 µL B5 buffer per well. Each wash is intercalated by a Duration00:05:00 aspiration dry.

5m
The waste container is then removed from under the binding plate which is dried again by a Duration00:10:00 aspriration

10m
An empty extraction plate is placed under the binding plate to retrieve the genomic DNA from it
DNA is eluted from the tissue binding plate with Amount100 µL BE buffer in each well and is collected in the new extraction plate underneath

After a Duration00:03:00 rest, the binding plate is dried for Duration00:02:00 and the elution is repeated with Amount100 µL BE buffer

5m
Retrieve the new extraction plate containing the genomic DNA and discard the rest