Apr 11, 2024

Public workspaceChloroform-free DNA Extraction - Ammonium Acetate Precipitation Method

DOI
dx.doi.org/10.17504/protocols.io.x54v9pekzg3e/v1
  • NERC Environmental Omics Facility (NEOF) Visitor Facility 1
  • 1University of Sheffield
NEOF - NERC Environmental Omics Facility
Open access
DOI: dx.doi.org/10.17504/protocols.io.x54v9pekzg3e/v1
Protocol Citation: NERC Environmental Omics Facility (NEOF) Visitor Facility 2024. Chloroform-free DNA Extraction - Ammonium Acetate Precipitation Method. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9pekzg3e/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 have been regularly using this method for decades.
Created: June 12, 2023
Last Modified: April 11, 2024
Protocol Integer ID: 83246
Abstract
A chloroform-free, cost-effective DNA extraction method for a variety of sample types.
Materials
Digsol: To make 500ml

  • 20ml 0.5M EDTA (pH 8.0)
  • 3.425g NaCl
  • 25ml 1M Tris-HCl (pH 8.0)
  • 430ml ddH2O

Autoclave then add 25ml of 20% SDS.


Low TE (Tris10mM,EDTA0.1mM): To make 500ml

  • 5ml 1M Tris-HCl (pH 8.0)
  • 100µl 0.5M EDTA (pH8.0)
  • 495ml ddH2O

Autoclave.
Protocol materials
ReagentProteinase K, 2mLQiagenCatalog #19131
Step 1
Reagent1M DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #43816
In 2 steps
ReagentAmmonium AcetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1542-500G
Step 4
Safety warnings
Attention
Use GLP and wear protective equipment. Avoid contact with skin.
All chemicals can be disposed of down the sink with copious amounts of water to dilute the ethanol down to >20% of the waste.

If inhaled: If unconscious, place in recovery position and seek medical advice. Keep the respiratory tract clear. If symptoms persist, call a physician.
In case of skin contact: Wash off immediately with soap and plenty of water while removing all contaminated clothes and shoes. If symptoms persist, call a physician.
In case of eye contact: Remove contact lenses. Protect unharmed eye. Rinse thoroughly with plenty of water for at least 15 minutes and consult a physician.
If swallowed: If accidentally swallowed, obtain immediate medical attention. Rinse mouth with water. Never give anything by mouth to an unconscious person.

Add Amount250 µL Digsol buffer (see Materials for recipe) and Amount10 µL ReagentProteinase K, 2mLQiagenCatalog #19131 to a lablelled 1.5ml tube.

1m
Tissue: Cut into small pieces (<1cm2) with a sterile razor blade on a sterile glass plate before adding to the 1.5mL tube.
Blood: Centrifuge blood sample at 13,000rpm for about 1 min (to pellet sample).
Remove sample from ethanol with toothpick and blot onto tissue. When almost dry, transfer the toothpick into the 1.5mL tube and jiggle to dislodge the blood. Remove toothpick and place in disinfectant.
Swab: Dry of ethanol and place into the 1.5mL tube. Snap off the end of the swap so the lid may close.
Feather: Cut the calamus of 1-3 feathers into small pieces with a sterile razor blade on a sterile glass plate before adding to the tube. If feathers are very small and blood spots are present on the tips, feathers can be added whole.
Hair: Place hair(s) into the 1.5mL tube preferably with the largest root and add Amount5 µL Reagent1M DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #43816
Insect: Degut and add to 1.5mL tube. If the insect is very small, starve for 48hrs before freezing. Add Amount5 µL Reagent1M DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #43816 . Crush with a pestle or add a metal lysing bead and place on TissueLyser (Qiagen) for 2min, 30/s.

5m
Vortex and place samples in a rotating oven at Temperature55 °C for Duration03:00:00 or DurationOvernight for maximum digestion.

3h
Add Amount300 µL 4MReagentAmmonium AcetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1542-500G .

1m
Vortex several times over a period of at least Duration00:15:00 at TemperatureRoom temperature .

15m
Centrifigation13000 rpm, 00:10:00

10m
Aspirate the supernatant (clear liquid containing the DNA) into a new labelled 1.5ml tube. Discard tube containing the pelleted protein debris.
1m
Add Amount1 mL 100% ethanol and invert the tube gently several times to precipitate DNA.

1m
Centrifigation13000 rpm, 00:10:00

10m
Pour off ethanol in a smooth motion, taking care not to lose the DNA pellet.
1m
Add Amount500 µL 70% ethanol and invert gently several times to clean the pellet.

1m
Centrifigation15000 rpm, 00:05:00

5m
Pour off ethanol in a smooth movement or using a pipette gently draw off the supernatant if fear of losing the pellet. Stand tubes upside-down on clean tissue until dry (approx. 30-60 minutes). This can be sped up by using the heat of a lamp from above.
30m
Once fully dry add approx. Amount100 µL Low TE (see Materials for recipe). Add less if a very tiny pellet or no pellet is observed. Flick sample to dislodge pellet.

1m
Place tubes in a thermomixer or shaking oven at Temperature50 °C for Duration00:30:00 to dissolve the pellet. If the pellet has not completely dissolved add more Low TE.

30m
Store at Temperature-20 °C (long term) or Temperature4 °C (short term).

Protocol references
https://onlinelibrary.wiley.com/doi/epdf/10.1046/j.0962-1083.2001.01355.x
Richardson DS, Jury FL, Blaakmeer K, Komdeur J, Burke T (2001) Parentage assignment and extra-group paternity in a cooperative breeder: the Seychelles warbler (Acrocephalus sechellensis). Molecular Ecology10, 2263-2273.

https://onlinelibrary.wiley.com/doi/abs/10.1034/j.1600-048X.2000.310208.x
The Evolution of Cooperative and Pair Breeding in Thornbills Acanthiza (Pardalotidae)
James A. Nicholls, Michael C. Double, David M. Rowell and Robert D. Magrath
Journal of Avian Biology
Vol. 31, No. 2 (Jun., 2000), pp. 165-176