Jan 28, 2022

Public workspaceDNA extraction from colonial tunicates

DOI
dx.doi.org/10.17504/protocols.io.b33sqqne
  • 1University of Fribourg
  • Blanchoud lab, UNIFR
Marta Wawrzyniak
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DOI: dx.doi.org/10.17504/protocols.io.b33sqqne
Protocol Citation: Marta Wawrzyniak, Simon Blanchoud 2022. DNA extraction from colonial tunicates. protocols.io https://dx.doi.org/10.17504/protocols.io.b33sqqne
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: January 20, 2022
Last Modified: January 28, 2022
Protocol Integer ID: 57170
Keywords: DNA extraction, colonial tunicates, ascidians
Abstract
This protocol has been successfully used with Botrylloides diegensis and was adapted to our needs based on the HotPhenol DNA extraction protocol.

Guidelines
Change gloves frequently, particularly as the protocol progresses from crude extracts to more purified materials. Use sterile tubes. Perform all steps on ice and use RNAse-free and DNase-free water unless otherwise stated.
Materials
Heat bath setup at 70 C (If working with fresh samples: glass beads 0.1mm and eppendorf thermal shaker)
phenol pH 8 (4 C)
Ph-Ch-IA: phenol:chloroform:isoamyl alcohol (25:24:1, best prepared fresh)
Ch-IA: chloroform:isoamyl alcohol (24:1)
SDS-Lysis buffer : 10mL Lysis buffer, 4mL SDS 10%
Lysis buffer 50mL: 12.3g 3M Sodium acetate (pH 5.2), 7.3g 0.5M EDTA , Nuclease-free water
12.3g 3M sodium acetate in 50mL Nuclease-free water
80% Ethanol
ultra pure water

This protocol was developed to extract both RNA and DNA in parallel (See RNA extraction from colonial tunicates , steps 1-7) using the same samples. However it could be run directly on fresh samples (steps 1.1-1.4).
Clean the slide from which you will take the colony of your interest. See Cleaning colonial ascidians.
Isolate a cleaned colony composed of approx. 20 zooids.
Transfer to a tube and spin at maximum speed for Duration00:02:00 .
2m
Remove the excess water.
Cell Lysis
Cell Lysis
4m 30s
4m 30s
Add Amount500 µL of Ph-Ch-IA solution and Amount350 µL of SDS-Lysis buffer.

Heat the tube at Temperature70 °C for Duration00:02:00 .
2m
If working with fresh samples, add glass beads 0.1mm to the tube and shake on eppendorf thermal shaker at 900rpm.
Mix by vortexing at maximum speed for Duration00:00:45 .

45s
Cool for Duration00:01:00 on ice.
1m
Mix again by vortexing at maximum speed for Duration00:00:45 .
45s
Heat the tubes at Temperature70 °C for Duration00:10:00 , mix regularly by inversion.
10m
If working with fresh samples, shake the tubes on eppendorf thermal shaker at 900rpm.
Mix again by vortexing at maximum speed for Duration00:00:45 .
45s
Cool for Duration00:01:00 on ice.
1m
Mix again by vortexing at maximum speed for Duration00:00:45 .
45s
DNA extraction
DNA extraction
3m
3m
Centrifuge at TemperatureRoom temperature at maximum speed for Duration00:03:00 .

3m
Transfer Amount400 µL of the upper aqueous phase to a new tube.

Add Amount400 µL of Ph-Cl-IA solution.

Shake the tube by inversion for Duration00:00:30 .

30s
Centrifuge at maximum speed for Duration00:03:00 .
3m
Transfer Amount300 µL of the upper aqueous phase to a new tube.
Add Amount300 µL of Ph-Cl-IA solution.
Shake the tube by inversion for Duration00:00:30 .
30s
Centrifuge at maximum speed for Duration00:03:00 .
3m
Transfer Amount200 µL of the upper aqueous phase to a new tube.
Add Amount200 µL of Cl-IA solution.
Shake the tube by inversion for Duration00:00:30 .
30s
Centrifuge at maximum speed for Duration00:03:00 .
3m
Transfer aqueous phase to a new tube.
DNA precipitation
DNA precipitation
3h 30m
3h 30m
Add 2 volumes of Concentration100 % volume Ethanol (typically Amount300-400 µL ).

Add 0.1 volume of Concentration3 Molarity (M) sodium acetate (typically Amount15-20 µL ).

Mix by inversion.
Incubate at Temperature-20 °C for Duration03:00:00 .

3h
Centrifuge at maximum speed for Duration00:20:00 at Temperature4 °C .

20m
Discard the supernatant.
Add Amount450 µL of cold Concentration80 % volume ethanol.

Centrifuge at maximum speed for Duration00:05:00 at TemperatureRoom temperature .
5m
Discard the supernatant.
Add Amount200 µL of cold Concentration80 % volume ethanol.
Centrifuge at maximum speed for Duration00:05:00 at TemperatureRoom temperature .
5m
Discard the supernatant.
Resuspend the pellet in ultra pure water (typically Amount20-100 µL ).

Measure the DNA concentration using the NanoDrop.
Store at Temperature-20 °C for short storage or at Temperature-80 °C for long storage.