Aug 22, 2024
CTAB Extraction from Sample Ground in Liquid Nitrogen
- 1Natural History Museum London
- Jordan Beasley: I have written up this protocol shared with me by Claire Griffin, Sequencing Assistant at the Natural History Museum, London.
- Darwin Tree of Life
Protocol Citation: Jordan Beasley 2024. CTAB Extraction from Sample Ground in Liquid Nitrogen. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw157zlmk/v1
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: April 29, 2024
Last Modified: August 22, 2024
Protocol Integer ID: 98938
Disclaimer
This protocol was shared with me by Claire Griffin, Sequencing Assistant at the Natural History Museum, London. Thank you Claire!
Abstract
This protocol describes the process of grinding a sample in liquid nitrogen and extracting DNA with a CTAB extraction.
Materials
1.5ml Eppendorf tubes
Pestle and mortar
Liquid nitrogen dewer
Liquid nitrogen
500µl CTAB
50µl Sarkosyl
50µl Proteinase K
200ul and 1ml tips
300µl Phenol chloroform
1ml SEVAC
400µl Isopropanol
500µl 70% Ethanol
Nuclease free water
Cryogenic Grinding and CTAB Extraction
Cryogenic Grinding and CTAB Extraction
Dispense some liquid nitrogen in a small dewer (small thermos size)
Fill a mortar with liquid nitrogen to cool it and let it boil off. When it has completely boiled away, add a small amount more into the mortar and grind with pestle to a fine powder. Scrape the powder into a 1.5ml tube.
Add 500µl CTAB, 50µl sarkosyl and 50µl Proteinase K to the powder and vortex to mix.
Place tube in a hot block at 60°C overnight or if in a rush 60-90 minutes.
After incubation add 300µl of phenol chloroform (This extracts the DNA into an aqueous layer).
Mix by inversion for 15 minutes on a rotator if you need high molecular weight. If you don't need high molecular weight, vortex well.
Centrifuge for 3 minutes at 16 rpm (max speed) and label a new tube.
Carefully remove the top aqueous layer and transfer to the new tube. Avoid disturbing any of the white protein layer.
Add 500µl SEVAC and mix by placing on a rotator for 15 minutes (or vortex well if you don't require high molecular weight DNA).
Centrifuge for 3 minutes at 16 rpm (max speed) and label a new tube.
Carefully remove the top aqueous layer and transfer to the new tube. Avoid disturbing any of the white protein layer.
Repeat steps 9-11.
Add 400µl isopropanol and mix by inverting - DO NOT VORTEX! You may see DNA precipitate out.
Centrifuge for 3 minutes at 16 rpm (max speed), you might see a pellet.
Remove as much of the liquid as you can without disturbing the pellet.
Add 500µl 70% ethanol (to wash away isopropanol)
Wash pellet by inverting several times
Centrifuge for 3 minutes at 16 rpm (max speed) and remove as much ethanol as possible without disturbing the pellet.
Place tube on a hot block with the lid open at 60°C for 5 minutes to evaporate off any remaining ethanol.
Resuspend in 30-50µl sterile water (flick the tube to help the pellet dissolve and if stubborn, pop back on the hot block for a few minutes).
Quantify extract on Nanodrop and/or Qubit.