The procedure is suitable for all types of tissues from wide variety of animal, blood and plant species. All DNA extraction steps are performed at weak acid pH (HEPES free acid) and optionally with hot chloroform for ‘difficult’ samples, and at room temperature.
The following protocol is designed for small and large tissue samples (tissue volume 100-200 μl).
Note that isolating genomic DNA not requires gentle mixing because the DNA not be sheared by vortexing.
CTAB method for DNA extraction protocol
CTAB solution: 1.5% CTAB, 1.5 M NaCl, 10 mM Na3EDTA, 0.1 M HEPES (pH ∼5.3);
100 ml: 1.5 g CTAB, 1.2 g HEPES-acid, 2 ml 0.5 M Na3EDTA, 30 ml 5 M NaCl;
Chloroform-isoamyl alcohol mix (24:1);
100% isopropanol (isopropyl alcohol, 2-propanol);
Fresh 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0).
2 ml Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball (5 or 6 mm) freeze at -80°C, grind in the MM300 Mixer Mill for 2 min at 30 Hz. Alternatively, grind the sample in lysis solution.
In 2 ml tube with mechanically disrupted seeds/leaves/herbarium or DNA solution (CTAB purification) add fresh 400 μl CTAB solution buffer with RNAse A (the sample mass should not exceed 50 mg), vortex very well and incubate the samples at 60-65°C during 30-60 min.
Add 2 volume 800 μl of chloroform, vortex very well (in the MM300 Mixer Mill for 1 min at 30 Hz); optionally: discard the lower phase (chloroform) contains dissolved polysaccharides.