Oct 12, 2022
CTAB DNA Extraction Protocol for Mollusks
- Lavanya Vythalingam1,
- Tim Regan1,
- Tim Bean1,
- Carolina Peñaloza1
- 1The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh
Protocol Citation: Lavanya Vythalingam, Tim Regan, Tim Bean, Carolina Peñaloza 2022. CTAB DNA Extraction Protocol for Mollusks. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwjpr2lmk/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: September 06, 2022
Last Modified: October 12, 2022
Protocol Integer ID: 69614
Keywords: comprehensive ctab extraction protocol, dna extraction of mollusk, dna extraction protocol, dna extraction, mollusk, extraction, dna
Abstract
This is a comprehensive CTAB extraction protocol intended for DNA extraction of mollusks. It includes detailed methodology and several reagent preparation.
Protocol materials
CTAB extraction solutionTeknovaCatalog #C2190
Proteinase K
Chloroform/Isoamyl Alcohol (24:1)Acros OrganicsCatalog #327155000
RNase A
Ethanol
Method
Slice tissue sample using a clean, sterile scalpel. Put sample into a 1.5 ml tube with 400 µL CTAB extraction solutionTeknovaCatalog #C2190 .
Add 10 µL Proteinase K 20 mg/mL and invert tube to mix.
Incubate at 50 °C for 60 minutes until lysed (or longer depending on tissue thickness). Invert tube occasionally to mix sample.
Add 400 µL Chloroform/Isoamyl Alcohol (24:1)Acros OrganicsCatalog #327155000 . Gently shake tube to emulsify sample.
Leave at room temperature for 2 minutes and shake tube to mix.
Spin tube at 13 000 rpm for 2 minutes and transfer upper layer to a 1.5 ml tube.
Add 5 µL ofRNase A 10 mg/mL . Leave for 30 minutes at 37 °C .
Add 1 equal volume of Chloroform/Isoamyl Alcohol (24:1)Acros OrganicsCatalog #327155000 .
Spin tube at 13 000 rpm for 2 minutes and transfer upper layer to a new 1.5 ml tube containing 900 µL CTAB dilution solution. Gently invert tube to mix. Leave in fridge overnight (optional).
Spin tube at 13 000 rpm for 10 minutes.
Discard supernatant with a micropipette and add 1000 µL 0.4 M NaCl in TE. Invert tube to wash.
Spin tube at 13 000 rpm for 5 minutes and remove supernatant.
Add 300 µL 1.42 M NaCl in TE to dissociate the CTAB from the DNA. Invert tube until pellet becomes transparent.
Note
May require gentle shaking at 37 °C
Add 600 µL Ethanol (previously left at -20 °C ). Invert manually until precipitation appears.
Note
Leave precipitating overnight for a higher DNA concentration
Centrifuge at full speed for 8 minutes, then remove supernatant. Dry at room temperature or in heat block at 37 °C
Resuspend in 50 µL EB buffer. Use lesser volume of EB buffer if a higher concentration is required.
Notes
All centrifugations were performed using a benchtop centrifuge (Eppendorf - 5415 D)
The Nucleic acid quality is measured using a Nanodrop, and the A260/A230 ratio of 1.8-2.0 indicates good DNA purity. Qubit can be used to determine nucleic acid concentration.
Store DNA sample at -20°C until further use.
Reagent preparation
CTAB Extraction Solution
Note
11.69 g Sodium ChlorideCatalog #PubChem CID: 5234
10 mL Tris HCl Buffer 1M Solution, Sterile pH 7.5Bio Basic Inc.Catalog #SD8124.SIZE.450ml
5 mL EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
3.33 g CTAB
Water to 100 mL
CTAB Dilution Solution
Note
10 mL Tris HCl Buffer 1M Solution, Sterile pH 7.5Bio Basic Inc.Catalog #SD8124.SIZE.450ml
4 mL EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
2 g CTAB
Water to 200 mL
0.4M Nacl in TE
Note
16 mL 5M NaCl
2 mL Tris HCl Buffer 1M Solution, Sterile pH 7.5Bio Basic Inc.Catalog #SD8124.SIZE.450ml
400 µL EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Water to 200 mL
1.42 M NaCl in TE
Note
24.8 mL 5MNaCl
1 mL Tris HCl Buffer 1M Solution, Sterile pH 7.5Bio Basic Inc.Catalog #SD8124.SIZE.450ml
200 µL EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Water to 100 mL