Dec 06, 2022
CTAB-based DNA extraction for citrus
- 1Division of Citrus Research, Institute of Fruit Tree and Tea Science, NARO
External link: https://doi.org/10.3389/fpls.2023.1163358
Protocol Citation: Shingo Goto 2022. CTAB-based DNA extraction for citrus. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpj8jpgzp/v1
Manuscript citation:
Goto S, Fujii H, Hamada H, Ohta S, Endo T, Shimizu T, Nonaka K, Shimada T, Allelic haplotype combinations at the region, including P-class pentatricopeptide repeat family genes, influence wide phenotypic variation in pollen grain number through a cytoplasmic male sterility model in citrus. Frontiers in Plant Science doi: 10.3389/fpls.2023.1163358
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 11, 2022
Last Modified: December 06, 2022
Protocol Integer ID: 69837
Keywords: CTAB, DNA extraction, citrus, High-salt precipitation solution
Abstract
CTAB-based protocols are used for genomic DNA extraction from many kinds of plant species. However, the protocols can’t necessarily completely remove contamination of polysaccharide and RNA in extracted genomic DNA solution. Especially, citrus leaves generally contain high polysaccharide. This protocol is a simple and efficient method for extracting genomic DNA for citrus without contamination of polysaccharide and RNA.
Buffer preparation
Buffer preparation
- 2×CTAB solution: 2% (w/v) CTAB, 100mMTris-HCl pH8.0, 1.4M NaCl, 20mM EDTA pH8.0 [1]
- High-salt precipitation solution: 1.2M NaCl , 0.8M Sodium citrate [2]
- 10 mg/ml RNase (Nippon gene)
- Chloroform
- Isopropanol
- 70% Ethanol
- TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0
Reference
- Allen GC, Flores-Vergara MA, Krasynanski S et al. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc 2006;1:2320–5.
- Chomczynski P, Mackey K. Modification of the TRI Reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 1995;19:492–5.
Homogenization and cell lysis
Homogenization and cell lysis
Preheat 2×CTAB solution to 60 °C in water bath. Add 2 % (v/v) of 2-Mercaptoethanol to the 2×CTAB solution just before use.
Homogenize 100 mg of fresh leaf in liquid Nitrogen. Add 800 µL of 2×CTAB solution and completely suspend homogenized leaf. Transfer the suspended solution to 2 ml tube.
Add4 µL of 10 mg/mL RNase, mix by inversion, and incubate at 37 °C for 00:15:00 (In order to prevent RNase contamination in laboratory, RNase treatment is conducted before denaturing proteins by chloroform).
Incubate at 56 °C for 00:30:00 inverting the tube once every 00:10:00 .
Chloroform extraction
Chloroform extraction
Add 300 µL of Chloroform and mix gently with tube rotator for 00:15:00 .
13000 rpm, 25°C, 00:10:00
Transfer supernatant carefully to new 2 ml tube.
Repeat step 6 and 7
Precipitation and wash of DNA pellet
Precipitation and wash of DNA pellet
Transfer 600 µL of supernatant to new 1.5 ml tube, add 300 µL of High-salt precipitation solution, and mix by inversion.
Add 300 µL of Isopropanol and mix by inversion.
15000 rpm, 25°C, 00:10:00
Discard and remove supernatant (DNA pellet is often transparent).
Add 1000 µL of 70% ethanol and mix by inversion 10 times to wash salts.
15000 rpm, 25°C, 00:05:00
Completely remove supernatant and dry up DNA pellet in air.
Dissolve pallet in 100 µL of TE buffer.
Result of gel electrophoresis
Result of gel electrophoresis
0.8% agarose gel electrophoresis
Lane 1-3: Genomic DNA extracted by isopropanol precipitation .
Lane 4: size marker
Lane 4-6: Genomic DNA extracted by isopropanol precipitation with high salt precipitation solution.
All genomic DNA is a citrus cultivar, 'Kiyomi'.