Sep 20, 2022
Version 3
CTAB genomic DNA extraction from Arabidopsis leaf material V.3
- Diep R Ganguly1,
- Pip Wilson2,
- Gonzalo Estavillo2,
- Xin Hou2,
- Barry Pogson2
- 1University of Pennsylvania;
- 2Australian National University
- Pogson Group
- EBL_ANU
Protocol Citation: Diep R Ganguly, Pip Wilson, Gonzalo Estavillo, Xin Hou, Barry Pogson 2022. CTAB genomic DNA extraction from Arabidopsis leaf material. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5y2dl1bz/v3Version created by Diep R Ganguly
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: April 22, 2021
Last Modified: September 20, 2022
Protocol Integer ID: 49315
Keywords: genomic DNA, Arabidopsis
Abstract
CTAB-based extraction of genomic DNA from Arabidopsis leaf tissue.
Guidelines
Gives reasonable quality and yield of gDNA. Ideal for DNA in PCRs applications, Dye-terminator Sanger sequencing reactions, and cloning. Can be used for next-generation sequencing applications, however, ensure you perform additional cleaning steps and longer centrifuge times. Run on a 1% agarose gel to ensure you have good quality and clean DNA preparation (and Nanodrop), often these preps yield a substantial portion of sheared nucleotides (likely RNA, so make sure you add RNase A during extraction).
Materials
MATERIALS
RNase AQiagenCatalog #19101
EDTA
1.5 mL Eppendorf tubes
water
Ethanol
NaClSigma AldrichCatalog #53014
Hexadecyltrimethylammonium bromideSigma AldrichCatalog #H6269
Tris-HCl (Tris-Hydrochloride), 100gmPromegaCatalog #H5121
2-Propanol (IsoPropanol)Bio Basic Inc.Catalog #PC8601.SIZE.4L
Tris-EDTA, pH 8.0AmbionCatalog #AM9849
ChloroformSigmaCatalog #366919-1L
Centrifuge
Water bath set to 65°C
Tissue lyser
1/8" steel ball bearings
Vortex
Centrifuge
RNase A (e.g. Promega #A7973 or Sigma #R6148)
Safety warnings
Perform chloroform steps in fume hood.
Before start
Ensure you grind your leaf tissue into a fine powder using mortar and pestle or Qiagen tissue lyser (place 1/8" steel ball bearing into tube with tissue sample).
Make sure leaf tissue remains frozen until the addition of CTAB buffer.
Cell lysis
Cell lysis
15m
15m
Prepare 2% CTAB buffer.
| A | B | |
| Reagent | [Cf] | |
| hexadecyltrimethylammonium bromide | 2% (w/v) | |
| NaCl | 1.4 M | |
| EDTA (pH 8) | 20 mM | |
| Tris-Cl (pH 8) | 100 mM |
2% CTAB buffer recipe
15m
Aliquot required volume of CTAB buffer and heat in water bath at 60 ºC for 5-10 minutes immediately before use.
10m
Add 300 µL / 100 mg leaf tissue of CTAB buffer.
2m
Add RNase A solution to a concentration 50-100 µg/mL.
2m
Mix well with a vortex. Invert samples by hand to ensure that all ground tissue is in solution.
5m
Incubate in water bath at 60 ºC for 30 - 60 minutes. Mix tubes periodically by inversion.
1h
Cool samples to room temperature .
10m
Phase separation
Phase separation
15m
15m
Add 300 µL chloroform and mix thoroughly with a vortex or vigorous shaking for 15 seconds.
5m
Centrifuge samples for 10 minutes at 20,000 rcf.
10m
Transfer upper aqueous (approx. 200 µL) phase to clean tube.
5m
Repeat steps 9-10 for a cleaner extract.
20m
Precipitation
Precipitation
15m
15m
Add equal volume of ice-cold 2-propanol and mix by inversion.
2m
Incubate for 30-60 min @ -20 °C
1h
Centrifuge for 15 min @ 20,000 rcf.
15m
Discard supernatant using pipette.
2m
Resuspend DNA
Resuspend DNA
17m
17m
Wash pellet with 1 mL of 70 % ethanol (mix by inversion).
2m
Centrifuge samples @ 9,200 rcf for 5 min.
5m
Remove as much ethanol as possible using a pipette, then allow pellet to air dry for 5 minutes.
10m
Resuspend gDNA in nuclease-free H2O or low EDTA TE buffer (10 mM Tris-Cl pH 8, 0.1 mM EDTA).
Test yield and purity of samples using a Nanodrop and running samples on a 1 % agarose gel.