May 17, 2018
- Ting Yang1,
- Chenyu Wu1
- 1BGI-Shenzhen
- GigaScience Press
- BGI
External link: https://doi.org/10.1093/gigascience/giz007
Protocol Citation: Ting Yang, Chenyu Wu 2018. DNA Extraction for plant samples by CTAB. protocols.io https://dx.doi.org/10.17504/protocols.io.pzqdp5w
Manuscript citation:
Huan Liu, Jinpu Wei, Ting Yang, Weixue Mu, Bo Song, Tuo Yang, Yuan Fu, Xuebing Wang, Guohai Hu, Wangsheng Li, Hongcheng Zhou, Yue Chang, Xiaoli Chen, Hongyun Chen, Le Cheng, Xuefei He, Hechen Cai, Xianchu Cai, Mei Wang, Yang Li, Sunil Kumar Sahu, Jinlong Yang, Yu Wang, Ranchang Mu, Jie Liu, Jianming Zhao, Ziheng Huang, Xun Xu, Xin Liu, Molecular digitization of a botanical garden: high-depth whole-genome sequencing of 689 vascular plant species from the Ruili Botanical Garden, GigaScience, Volume 8, Issue 4, April 2019, giz007, https://doi.org/10.1093/gigascience/giz007
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: May 09, 2018
Last Modified: May 22, 2018
Protocol Integer ID: 12048
Abstract
DNA extraction of −80◦C stored leaves by CTAB.
Materials
MATERIALS
PVPMerck MilliporeSigma (Sigma-Aldrich)
20 mM EDTAMerck MilliporeSigma (Sigma-Aldrich)
100 mM Tris-HClMerck MilliporeSigma (Sigma-Aldrich)
1.5 M NaClMerck MilliporeSigma (Sigma-Aldrich)
2% CTABMerck MilliporeSigma (Sigma-Aldrich)
1% β-mercaptoethanolBBI Biotech
Chloroform: Isoamyl alcoholSangon Biotech
isopropanolSangon Biotech
sodium acetateQiagen
ethanolBBI Biotech
TE bufferThermo Fisher Scientific
STEP MATERIALS
PVPMerck MilliporeSigma (Sigma-Aldrich)
20 mM EDTAMerck MilliporeSigma (Sigma-Aldrich)
100 mM Tris-HClMerck MilliporeSigma (Sigma-Aldrich)
1.5 M NaClMerck MilliporeSigma (Sigma-Aldrich)
2% CTABMerck MilliporeSigma (Sigma-Aldrich)
1% β-mercaptoethanolBBI Biotech
Chloroform: Isoamyl alcoholSangon Biotech
isopropanolSangon Biotech
sodium acetateQiagen
ethanolBBI Biotech
TE bufferThermo Fisher Scientific
PVPMerck MilliporeSigma (Sigma-Aldrich)
20 mM EDTAMerck MilliporeSigma (Sigma-Aldrich)
100 mM Tris-HClMerck MilliporeSigma (Sigma-Aldrich)
1.5 M NaClMerck MilliporeSigma (Sigma-Aldrich)
2% CTABMerck MilliporeSigma (Sigma-Aldrich)
1% β-mercaptoethanolBBI Biotech
Chloroform: Isoamyl alcoholSangon Biotech
isopropanolSangon Biotech
sodium acetateQiagen
ethanolBBI Biotech
TE bufferThermo Fisher Scientific
Protocol materials
100 mM Tris-HClMerck MilliporeSigma (Sigma-Aldrich)
1% β-mercaptoethanolBBI Biotech
isopropanolSangon Biotech
ethanolBBI Biotech
2% CTABMerck MilliporeSigma (Sigma-Aldrich)
20 mM EDTAMerck MilliporeSigma (Sigma-Aldrich)
1% β-mercaptoethanolBBI Biotech
TE bufferThermo Fisher Scientific
PVPMerck MilliporeSigma (Sigma-Aldrich)
1.5 M NaClMerck MilliporeSigma (Sigma-Aldrich)
sodium acetateQiagen
100 mM Tris-HClMerck MilliporeSigma (Sigma-Aldrich)
ethanolBBI Biotech
sodium acetateQiagen
PVPMerck MilliporeSigma (Sigma-Aldrich)
TE bufferThermo Fisher Scientific
isopropanolSangon Biotech
TE bufferThermo Fisher Scientific
PVPMerck MilliporeSigma (Sigma-Aldrich)
1% β-mercaptoethanolBBI Biotech
Chloroform: Isoamyl alcoholSangon Biotech
1.5 M NaClMerck MilliporeSigma (Sigma-Aldrich)
sodium acetateQiagen
2% CTABMerck MilliporeSigma (Sigma-Aldrich)
Chloroform: Isoamyl alcoholSangon Biotech
20 mM EDTAMerck MilliporeSigma (Sigma-Aldrich)
100 mM Tris-HClMerck MilliporeSigma (Sigma-Aldrich)
ethanolBBI Biotech
Chloroform: Isoamyl alcoholSangon Biotech
2% CTABMerck MilliporeSigma (Sigma-Aldrich)
20 mM EDTAMerck MilliporeSigma (Sigma-Aldrich)
isopropanolSangon Biotech
1.5 M NaClMerck MilliporeSigma (Sigma-Aldrich)
PVPMerck MilliporeSigma (Sigma-Aldrich)
20 mM EDTAMerck MilliporeSigma (Sigma-Aldrich)
100 mM Tris-HClMerck MilliporeSigma (Sigma-Aldrich)
1.5 M NaClMerck MilliporeSigma (Sigma-Aldrich)
2% CTABMerck MilliporeSigma (Sigma-Aldrich)
1% β-mercaptoethanolBBI Biotech
Chloroform: Isoamyl alcoholSangon Biotech
isopropanolSangon Biotech
sodium acetateQiagen
ethanolBBI Biotech
TE bufferThermo Fisher Scientific
Grind −80◦C stored leaves (100-200 mg) to fine powder using liquid nitrogen in the presence of 50 mg PVP (PolyVinyl Pyrrolidone, Mr 10,000). Note: It' s very important that the tissue doesn't thaw.
PVPSigma Aldrich
Add 1.5 mL of freshly prepared 2x CTAB buffer and incubate at 60◦C for 45 min with intermittent shaking every 10 minutes.
20 mM EDTA (PH 8.0, 0.5M) 40 ml
100 mM Tris-HCl (PH 8.0, 1M) 100 ml
1.5 M NaCl 87.6 g
2% CTAB (w/v) 20 g
1% β-mercaptoethanol 10 ml (add before use)
Then add distilled water to make it up to 1000 ml.
00:45:00
20 mM EDTASigma Aldrich
100 mM Tris-HClSigma Aldrich
1.5 M NaClSigma Aldrich
2% CTABSigma Aldrich
1% β-mercaptoethanolSigma Aldrich
Centrifuge at 12,000 rpm for 15 min at room temperature.
00:15:00
Carefully transfer the aqueous phase into a new tube. Note: Use wide-bore tips for transferring the aqueous phase to avoid mechanical damage to DNA.
Add double volume of Chloroform: Isoamyl alcohol (24 : 1) under fume hood, and invert gently 15 to 20 times and centrifuge at 12,500 rpm for 15 min. Note: If the aqueous layer appears translucent, repeat the stepuntil the solution is transparent. Be careful to avoid transferring any chloroform.
00:15:00
Chloroform: Isoamyl alcoholSigma Aldrich
Add double volume of chilled isopropanol followed by 1/3rd volume of 3 M sodium acetate (pH 5.2) and keep at -20◦C for one hour to precipitate the DNA. Note: The longer the chilled incubation, the more the precipitation.
01:00:00
isopropanolSigma Aldrich
sodium acetateSigma Aldrich
Centrifuge at 12,000 rpm for 15 min and discard the supernatant.
00:15:00
To the pellet, add 70% chilled ethanol and spool out the pellet carefully and centrifuge again at 12,000 rpm for 15 min, and repeat the step again.
00:15:00
ethanolSigma Aldrich
Discard the supernatant and vacuum dry or air dry the pellet at room temperature. Note: Make sure that there is no residual ethanol, this is very critical especially if the DNA is to be used directly for PCR. Overdrying should also be avoided as it makes the pellet difficult to resuspend.
Add 40-50 μL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8), and quantify the DNA using Qubit flurometer/Nanodrop, and run an 0.8% Agarose gel electrophoresis.
TE bufferSigma Aldrich
Store at -20◦C/-40◦C till further use.