DNA extraction from colonial tunicates
Marta Wawrzyniak, Simon Blanchoud
Abstract
This protocol has been successfully used with Botrylloides diegensis and was adapted to our needs based on the HotPhenol DNA extraction protocol.
Steps
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 0h 2m 0s .
Remove the excess water.
Cell Lysis
Add 500µL of Ph-Ch-IA solution and 350µL of SDS-Lysis buffer.
Heat the tube at 70°C for 0h 2m 0s .
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 0h 0m 45s .
Cool for 0h 1m 0son ice.
Mix again by vortexing at maximum speed for 0h 0m 45s.
Heat the tubes at 70°C for 0h 10m 0s, mix regularly by inversion.
If working with fresh samples, shake the tubes on eppendorf thermal shaker at 900rpm.
Mix again by vortexing at maximum speed for 0h 0m 45s.
Cool for 0h 1m 0son ice.
Mix again by vortexing at maximum speed for 0h 0m 45s.
DNA extraction
Centrifuge at Room temperature at maximum speed for 0h 3m 0s .
Transfer 400µL of the upper aqueous phase to a new tube.
Add 400µL of Ph-Cl-IA solution.
Shake the tube by inversion for 0h 0m 30s .
Centrifuge at maximum speed for 0h 3m 0s .
Transfer 300µL of the upper aqueous phase to a new tube.
Add 300µL of Ph-Cl-IA solution.
Shake the tube by inversion for 0h 0m 30s .
Centrifuge at maximum speed for 0h 3m 0s .
Transfer 200µL of the upper aqueous phase to a new tube.
Add 200µL of Cl-IA solution.
Shake the tube by inversion for 0h 0m 30s .
Centrifuge at maximum speed for 0h 3m 0s .
Transfer aqueous phase to a new tube.
DNA precipitation
Add 2 volumes of 100% volume Ethanol (typically 300-400µL ).
Add 0.1 volume of 3Molarity (M) sodium acetate (typically 15-20µL ).
Mix by inversion.
Incubate at -20°C for 3h 0m 0s .
Centrifuge at maximum speed for 0h 20m 0s at 4°C .
Discard the supernatant.
Add 450µL of cold 80% volume ethanol.
Centrifuge at maximum speed for 0h 5m 0s at 4Room temperature .
Discard the supernatant.
Add 200µL of cold 80% volume ethanol.
Centrifuge at maximum speed for 0h 5m 0s at 4Room temperature .
Discard the supernatant.
Resuspend the pellet in ultra pure water (typically 20-100µL ).
Measure the DNA concentration using the NanoDrop.
Store at -20°C for short storage or at -80°C for long storage.
