High Molecular Weight DNA extraction from tunicates

Clean the slide from which you will take the colony of your interest. See Cleaning colonial ascidians.

Isolate a cleaned colony composed of approx. 20-30 zooids.

Transfer to a 2 mL tube and spin at maximum speed for 0h 2m 0s .

Remove the excess water.

Homogenize the sample in500µL of TE buffer using a sterile plastic pestle.

Add 500µL of SDS and 4µL of RNase and mix by vortexing.

Incubate at 55°C for 0h 10m 0s .

Add 4µL of Proteinase K and mix by vortexing.

Incubate at 55°C for 0h 15m 0s .

Add 1mL of phenol and mix well by inverting the tube until the phases are completely mixed.

Spin at 10000rcf,0h 0m 0s for 0h 5m 0s and carefully transfer the 500µL of the upper phase to a new 2 mL tube.

Add 500µL of (1:1 v/v) phenol/chloroform and mix well by inverting the tube.

Spin at 10000rcf,0h 0m 0s for 0h 5m 0s and carefully collect the 300µL of upper phase to a new 2 mL tube.

Precipitate the DNA by addition of 30µL 3 M sodium acetate and 600µL of ethanol. Mix gently by inverting the tube.

Spin at 10000rcf,0h 0m 0s for 0h 3m 0s to pellet nucleic acids and carefully remove and discard supernatant.

Wash in 1mL cold ethanol (-20°C ) and invert gently several times.

Spin at 10000rcf,0h 0m 0s for 0h 2m 0s .

Carefully remove and discard supernatant and place the tube up-side-down on a paper towel for 0h 10m 0s .

Resuspend the pellet gently in RNase-free water at 37°C for 1h 0m 0s .

Quantify the DNA concentration and quality.

Store at -20°C or -80°C (for longer storage).