Feb 22, 2022

Public workspaceHigh Molecular Weight DNA extraction from tunicates

DOI
dx.doi.org/10.17504/protocols.io.b5axq2fn
  • 1University of Fribourg
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DOI: dx.doi.org/10.17504/protocols.io.b5axq2fn
Protocol Citation: Marta Wawrzyniak, Simon Blanchoud 2022. High Molecular Weight DNA extraction from tunicates . protocols.io https://dx.doi.org/10.17504/protocols.io.b5axq2fn
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: February 18, 2022
Last Modified: February 22, 2022
Protocol Integer ID: 58423
Keywords: high molecular weight DNA, tunicates, ascidians, DNA extraction
Abstract
This protocol has been successfully used with Botrylloides diegensis and has been based on the following publication (with small changes):

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793(89)80446-6

Guidelines
from step 8 on, avoid vortexing or vigorous shaking; invert the tube gently to avoid breaking the DNA.
Materials
TE Buffer: 100 mM Tris-borate, pH 8.0 + 50 mM Na2EDTA
14 % (w/v) SDS (sodium dodecyl sulfate)
10 mg/mL RNase
20 mg/mL Proteinase K
Phenol pH 8.0
Chloroform
1:1 (v/v) Phenol pH 8.0/Chloroform
Cold ethanol (-20 °C)
3M sodium acetate
RNase-free water
2mL tubes
sterile plastic pestle


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 Duration00:02:00 .
2m
Remove the excess water.
Homogenize the sample inAmount500 µL of TE buffer using a sterile plastic pestle.
Add Amount500 µL of SDS and Amount4 µL of RNase and mix by vortexing.

Incubate at Temperature55 °C for Duration00:10:00 .

10m
Add Amount4 µL of Proteinase K and mix by vortexing.

Incubate at Temperature55 °C for Duration00:15:00 .
15m
Add Amount1 mL of phenol and mix well by inverting the tube until the phases are completely mixed.

Spin at Centrifigation10000 rcf for Duration00:05:00 and carefully transfer the Amount500 µL of the upper phase to a new 2 mL tube.


5m
Add Amount500 µL of (1:1 v/v) phenol/chloroform and mix well by inverting the tube.

Spin at Centrifigation10000 rcf for Duration00:05:00 and carefully collect the Amount300 µL of upper phase to a new 2 mL tube.

5m
Precipitate the DNA by addition of Amount30 µL 3 M sodium acetate and Amount600 µL of ethanol. Mix gently by inverting the tube.

Spin at Centrifigation10000 rcf for Duration00:03:00 to pellet nucleic acids and carefully remove and discard supernatant.

3m
Wash in Amount1 mL cold ethanol (Temperature-20 °C ) and invert gently several times.

Spin at Centrifigation10000 rcf for Duration00:02:00 .
2m
Carefully remove and discard supernatant and place the tube up-side-down on a paper towel for Duration00:10:00 .

10m
Resuspend the pellet gently in RNase-free water at Temperature37 °C for Duration01:00:00 .

1h
Quantify the DNA concentration and quality.
Store at Temperature-20 °C or Temperature-80 °C (for longer storage).