High-throughput mini DNA extraction protocol
Supplies & Solutions:
Extraction Buffer [CTAB (2-3% w/v), 1.4 M NaCl, 20 mM EDTA, 100mM
Tris-HCl (pH-8)], 0.2% - mercaptoethanol( optional),Chloroformisoamyl alcohol (24:1), isopropanol, Absolute Ethanol, 70% Ethanol,
TE buffers (1 mM Tris, 0.1mM EDTA & 10 mM Tris, 1mM EDTA [pH
8]) and 3M Sodium acetate(NaOAc) [pH 5.2)
A. Sample preparation
• Harvest leaves from seedlings 10-15 days after sowing.
• Place ~ 50 mg of leaf tissue in 12 x 8-well strip tube with strip caps
(ThermoFisher, USA) in a 96 deep-well plate together with two 5/32”
stainless steel grinding balls
B. CTAB extraction
• Add 450 μl of preheated (65°C) extraction buffer [100 mM Tris-HCl
(pH- 8), 1.4 M NaCl, 20 mM EDTA, CTAB (2-3% w/v) to each sample and
secure with 8-strip caps.
• Grind samples in a Geno Grinder 2000 (Spex CertiPrep, USA), following
the manufacturer’s instructions, at 1200 strokes/min for 2-3 times at 2
min interval.
• Incubate for 30 min in a 65°C water bath with occasional mixing.
C. Solvent extraction
• Add 450 μl of chloroform-isoamylalcohol (24:1) to each sample and
invert twice to mix.
• Centrifuge at 5500 rpm for 10 min (Sigma centrifuge model 4K15C)
• Transfer aqueous layer to fresh strip tubes
D. DNA pellet precipitation and RNase treatment
• Add 0.7 vol of isopropanol (stored at -20°C) to each sample and invert
once to mix.
• Centrifuge the samples at 5500 rpm for 15 min.
• Decant supernatant from each sample and air dry the pellet for 30 min.
• Alternatively use Speev –Vac for drying for 10-12 min
• Add 200 μl TE (1 mM Tris, 0.1mM EDTA [pH 8]) and 3 μl RNAse A (10
mg/ mL) to each sample.
E. Solvent extraction and purification
• Add 200 μl phenol-chloroform-isoamylalcohol (25:24:1) to each sample
and invert twice to mix.
• Centrifuge the samples at 5000 rpm for 5 min.
• Transfer the aqueous layer to fresh strip tubes
• Add 200 μl chloroform-isoamylalcohol (24:1) to each sample and invert
twice to mix.
• Centrifuge the samples at 5000 rpm for 5 min.
• Transfer the aqueous layer to fresh strip tubes
• Add twice the volume of absolute ethanol and 1/10 volume of 3M
NaOAc [pH 5.2) to each sample and place in -20°C for 5 min.
• Centrifuge at 5500 rpm for 15 min.
• Decant supernatant from each sample and add 200 μl of 70% ethanol
and centrifuge at 5500 rpm for 5 min
• Decant supernatant and air dry the pellet for approximately 1 hour or
use Speed- Vac for drying
• Resuspend pellet in 100 μl TE (10 mM Tris, 1mM EDTA [pH 8]) and
store at 4°C for immediate use or at-20°C for long-term use.