Oct 29, 2024
Modified CTAB procedure for extraction of plant DNA from bird feces
This protocol is a draft, published without a DOI.
- Carina Motta1,
- Vinicius Marciano de Godoy1,
- Marina Corrêa Côrtes1
- 1UNESP
- Metabarcoding of Plant DNA in Bird Feces
Protocol Citation: Carina Motta, Vinicius Marciano de Godoy, Marina Corrêa Côrtes 2024. Modified CTAB procedure for extraction of plant DNA from bird feces . Protocol exchange https://protocols.io/view/modified-ctab-procedure-for-extraction-of-plant-dn-dqd95s96
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: October 23, 2024
Last Modified: October 29, 2024
Protocol Integer ID: 110753
Keywords: Metabarcoding, DNA extraction, bird feces
Funders Acknowledgement:
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Grant ID: 2021/03467-3
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Grant ID: 2018/19011-6
Abstract
Modified CTAB procedure to extract plant DNA from bird feces stored in alcohol. Final concentration of the 2% CTAB solution include the following: 2 % volume CTAB, 1.4 Molarity (M) NaCl, 20 millimolar (mM) EDTA, 100 millimolar (mM) Tris-HCl (pH 8.0), 2 % volume PVP (40). We recommend making a new batch of CTAB solution before each extraction. Modifications include an increased lysis time (up to 12 hours), as well as the use of sodium acetate (3 Molarity (M) ) and NaCl (6 Molarity (M) ) in the wash step. DNA acquired is suitable for PCR.
Guidelines
A fume hood is required to carry out this protocol due to the use of chloroform (CIA 1:24).
Materials
Find a list of materials required per sample below:
Equipment
Analytical balance
Dry bath
Centrifuge
Mini Beadbeater-96
10 beads
1000 μL pipette
100 μL pipette
10 μL pipette
Solutions
800 μL extraction buffer (2% CTAB)
1200 μL of CIA (chloroform-isoamyl alcohol) 24:1
300 μL of 6M NaCl
60 μL of 3M sodium acetate
400 μL cold isopropanol (-20°C)
2 mL of 70% ethanol
1 mL of 95% (or absolute) ethanol
49.5 μL TE
0.5 μL RNase
Distilled water
Disposables
2 tubes (2 mL)
1 tube (1.5 mL)
Tips
Sample
50 to 100 mg of fecal material
200 μL of the alcohol in which the feces were preserved
Safety warnings
Chloroform toxic if swallowed or inhaled. It can cause severe and irreversible health effects, including death. Short-term exposure to high levels of chloroform can damage the central nervous system, liver, and kidneys. Long-term exposure to lower levels of chloroform can damage the liver and kidneys. Use appropriate PPE when applying this protocol.
Before start
This protocol is intended to be carried out over the course of two days. We recommend preparing your samples in the afternoon/evening and carrying out the extraction the following day. Samples will need to stay an additional night in the refrigerator to elute, although you can use a dry bath to accelerate this process.
Prepare materials and samples for extraction (Day 1)
Prepare materials and samples for extraction (Day 1)
Preparation of Materials
- Verify that all solutions are prepared and autoclaved material is available
- Turn on the dry bath to 65°C
- Prepare three identical sets of numbered tubes (2 sets of 2 mL tubes and 1 set of 1.5 mL tubes)
- Record in the lab notebook the correspondence between the tube numbers and sample IDs
Preparation of Samples
Weigh 50 to 100 mg (~80 mg) of fecal material for each sample directly into the tube. Add 200 μL of the sample alcohol to the separated feces.
Place the open tubes in the dry bath (65°C) for ~2 hours until the alcohol evaporates.
Add 800 μL of CTAB buffer and 20 μL of Proteinase K with 10 porcelain beads. Place in the Minibeater for 5 minutes.
Incubate the tubes in a water bath at 60°C overnight (~12 hours)
Extraction (Day 2)
Extraction (Day 2)
Extraction with CIA I
Let samples come to room temperature. In a fume hood, perform the first extraction with an organic solvent by adding 600 μL of CIA (chloroform-isoamyl alcohol 24:1).
Vortex the tubes and then invert them for 5 minutes (at least 20 times) or until a homogeneous emulsion forms.
Centrifuge the tubes in a microcentrifuge at maximum speed (13,000 rpm) for 10 minutes.
Carefully remove the tubes from the centrifuge, avoiding disturbance of the interface between the two phases.
Pipette the upper (aqueous) phase into a new tube.
- To expedite this step, set the pipette to 180 μL and withdraw four fixed aliquots (~720 μL). Even if some volume is left behind, this helps avoid contamination with the lower organic phase.
Extraction with CIA II
Repeat the extraction with 600 μL of CIA (steps 3/4/5).
In a fume hood, perform the second extraction with an organic solvent by adding 600 μL of CIA (chloroform-isoamyl alcohol 24:1).
Vortex the tubes and then invert them for 5 minutes (at least 20 times) or until a homogeneous emulsion forms.
Centrifuge the tubes in a microcentrifuge at maximum speed (13,000 rpm) for 10 minutes.
Carefully remove the tubes from the centrifuge, avoiding disturbance of the interface between the two phases.
Pipette the upper (aqueous) phase into a new tube
- To expedite this step, set the pipette to 180 μL and withdraw three fixed aliquots (~540 μL). Even if some volume is left behind, this helps avoid contamination with the lower organic phase
Precipitate DNA
Add ½ volume of 6M NaCl (~270 μL) and 1/10 volume (54 μL) of 3M sodium acetate. Then add 2/3 of the volume of the aqueous solution (~400 μL) of cold isopropanol (-20°C).
- Mix gently to precipitate the nucleic acids.
Place the tubes in a -20°C freezer for 2 hours.
Wash DNA
Centrifuge the tubes at 13,000 rpm in a microcentrifuge for 10 minutes to form a pellet.
- Don’t worry if you can't see the pellet—it’s likely there, but it may be small and hard to see.
Carefully pour off as much supernatant as possible without losing the pellet.
Wash the pellet with 500 μL of 70% ethanol.
Let the pellet sit immersed for 5 minutes.
Centrifuge for 5 minutes at 13,000 rpm.
Wash the pellet with 500 μL of 100% ethanol.
Let the pellet sit immersed for 2 minutes.
Centrifuge for 2 minutes at 13,000 rpm.
Elute DNA
Resuspend the pellet in 50 μL of TE with RNase.
Mix 49.5 μL of TE with 0.5 μL of RNase, and multiply by the number of samples.
Store in freezer at -20°C