Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials
2.2. Genomic DNA Extraction
2.2.1. CTAB-Based Method
2.2.2. Qiagen Method
2.2.3. Optimization of Nuclear Pellet Method
Reagents
- Solution I: 15% sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0), and 500 mM NaCl
- Resuspension buffer: 20 mM Tris-HCl (pH 8.0) and 10 mM EDTA (pH 8.0)
- 20% sodium dodecyl sulfate (SDS)
- 7.5 M ammonium acetate
- 5 M NaCl
- Isopropanol
- TE buffer: 10 mM Tris-HCl (pH 7.6) and 0.1 mM EDTA
- RNase A: a working solution of 1 µg/µL (final concentration ≈ 10 ng/µL)
- Proteinase K: a working solution of 2 µg/µL (final concentration ≈ 80 ng/µL)
- PCI: phenol/chloroform/isoamyl alcohol (25:24:1)
- CIA: chloroform/isoamyl alcohol (24:1)
- 100% ethanol (v/v)
- 70% ethanol (v/v)
- QubitTM dsDNA HS Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA)
Equipment
- Mortar and pestle
- Dry block heater
- Table-top high-speed microcentrifuge
- Freezer (−20 °C)
- Gel electrophoresis system
- Eppendorf BioSpectrometer® fluorescence (Eppendorf AG, Hamburg, Germany)
Protocol
- Weigh 1 g of young fresh leaf tissue and cut into small pieces using clean and sharp scissors. Place the cut tissue in liquid nitrogen and grind thoroughly with a mortar and pestle. Add 12 mL of Solution I to the fine tissue powder and suspend well. Transfer the suspended tissue (2 mL in each) into six individual 2 mL microcentrifuge tubes.
- Centrifuge the tubes containing the suspended tissue at 500 rpm for 3 min at 4 °C. Discard the upper phase carefully. Centrifuge at 1000 rpm for 1 min at 4 °C and discard the upper phase.
- Add 450 µL of resuspension buffer and 30 µL of 20% SDS into each tube containing tissue, shake briefly, and incubate at 70 °C for 15 min.
- Add 230 µL of 7.5 M NH4OAc into each tube, shake vigorously, and incubate the reaction mixture on ice for at least 30 min.
- Centrifuge the reaction mixture at 15,000 rpm for 20 min at 4 °C and divide equal amounts of the cleared supernatant into six individual microcentrifuge tubes, labeled #1–6. Repeat this step (once).
- Add 1 volume of isopropanol to the supernatant of each tube, gently mix by inverting, and centrifuge at 15,000 rpm for 15 min at 25 °C. Discard the supernatant and air-dry pellet for 5–10 min (do not excess dry).
- Add 50 µL of TE buffer individually to tubes #1–5 and wait until the pellet has dissolved. Transfer the dissolved DNA from tubes #1–5 into tube #6. Add 50 µL TE buffer to the original tube #1, allow to dissolve, then transfer it to tube #2. Repeat this transfer and dissolving process until tube #5, then transfer the DNA to tube #6. The final volume of the DNA sample in tube #6 will be 300 µL.
- Add 600 µL of 100% ethanol to the sample in tube #6, centrifuge at 15,000 rpm for 10 min at 25 °C, and decant the supernatant. Again, add 600 µL of 100% ethanol to the DNA pellet in tube #6, centrifuge at 15,000 rpm for 1 min, and decant the supernatant.
- Air-dry the pellet for 5–10 min. Resuspend the pellet in 50 µL of TE buffer (do not vortex).
- Add 0.5 µL of RNase A to the solution and incubate at 37 °C for 1 h.
- Add 2.2 µL of proteinase K to the solution and incubate at 37 °C for 1 h.
- Check gDNA quality by electrophoresis using a 0.7% agarose gel (optional).
- Add 400 µL of TE buffer to the digested sample containing the DNA and mix gently by pipetting.
- Add 450 µL of PCI to the DNA solution, gently mix, and centrifuge at 15,000 rpm for 15 min at 4 °C. Transfer the cleared supernatant to a new 1.5 mL collection tube. Repeat this step (once).
- Add 450 µL of CIA to the DNA solution, mix gently, and centrifuge at 15,000 rpm for 15 min at 4 °C. Transfer the cleared supernatant to a new 1.5 mL collection tube. Repeat this step (once).
- Add 27 µL of 5 M NaCl and 1 mL of 100% ethanol to the solution, gently mix, and incubate at −20 °C for 1 h.
- Centrifuge at 15,000 rpm for 15 min at 4 °C and discard the supernatant.
- Wash the pellet with 100 µL of 70% ethanol, centrifuge at 15,000 rpm for 5 min, and discard the ethanol.
- Wash the pellet with 100 µL of 100% ethanol, centrifuge at 15,000 rpm for 5 min, and discard the supernatant. Again, add 100 µL of 100% ethanol to the pellet and wash and discard the ethanol.
- Air-dry the pellet for 5–10 min. Resuspend the pellet in 11 µL of TE buffer.
- Dilute the DNA with TE buffer (as required) for downstream analysis.
2.3. DNA Quantification
2.4. DNA Quality Assessment
2.5. Quantification of Nuclear, Chloroplast, and Mitochondrial DNAs by qPCR
3. Results and Discussion
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Experimental Method | A260/A280 Ratio | A260/A230 Ratio | DNA Yield (µg/g Fresh Tissue) |
---|---|---|---|
CTAB-based | 2.06 ± 0.10 | 2.39 ± 0.04 | 6.59 ± 0.80 c |
QIAGEN DNeasy Plant Mini Kit | 1.91 ± 0.03 | 2.31 ± 0.03 | 9.03 ± 1.50 b |
Nuclear pellet | 2.08 ± 0.01 | 2.46 ± 0.02 | 11.78 ± 1.38 a |
Optimized nuclear pellet | 1.87 ± 0.02 | 2.26 ± 0.05 | 10.40 ± 1.32 ab |
Sample Name | DNA Submitted (ng/µL TE Buffer) | Total DNA Submitted (µg) | NGS Quality Control Results |
---|---|---|---|
Yukinko-mai | 330.7 | 5.29 | Passed |
Introgressed line (BC3F2) | 256.8 | 4.11 | Passed |
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Rana, M.M.; Aycan, M.; Takamatsu, T.; Kaneko, K.; Mitsui, T.; Itoh, K. Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue. Methods Protoc. 2019, 2, 54. https://doi.org/10.3390/mps2020054
Rana MM, Aycan M, Takamatsu T, Kaneko K, Mitsui T, Itoh K. Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue. Methods and Protocols. 2019; 2(2):54. https://doi.org/10.3390/mps2020054
Chicago/Turabian StyleRana, Md Masud, Murat Aycan, Takeshi Takamatsu, Kentaro Kaneko, Toshiaki Mitsui, and Kimiko Itoh. 2019. "Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue" Methods and Protocols 2, no. 2: 54. https://doi.org/10.3390/mps2020054