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Total DNA isolation protocol

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: 2% CTAB, 2 M NaCl, 10 mM Na3EDTA (pH ∼5.3);
    100 ml: 2 g CTAB, 1.2 g HEPES-acid, 2 ml 0.5 M Na3EDTA, 40 ml 5 M NaCl;
  • Chloroform-isoamyl alcohol mix (24:1);
  • 100% isopropanol (isopropyl alcohol, 2-propanol);
  • 70% ethanol;
  • Fresh 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0).
  1. 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.
  2. In 2 ml tube with mechanically disrupted seeds or leaves or herbarium or blood or DNA solution (CTAB purification) add fresh 400 μl CTAB solution buffer with RNAse A (the sample volume should not exceed 5% of lysis buffer), vortex very well and incubate the samples at 60-65°C during 30-60 min.
  3. Add 2 volume (0.8 ml) of chloroform, vortex very well (in the MM300 Mixer Mill for 1 min at 30 Hz); optionally: incubate the samples at 60-65°C during 30 min.
  4. Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the upper aqueous layer to a new 2 ml microcentrifuge tube.
  5. Optionally: add 0.8 ml of chloroform, vortex very well for 1 minute creating an emulsion (in the MM300 Mixer Mill at 30 Hz).
  6. Spin at maximum speed in a microcentrifuge for 5 minutes.
  7. Transferred the upper aqueous layer to a new 1.5 ml microcentrifuge tube which contains of 400 μl 2-propanol (or 1 ml 96% ethanol), vortex well and centrifuge the tubes at maximum speed in a microcentrifuge for 3 minutes.
  8. Discard supernatant and wash pellet by adding 1 ml 70% EtOH, vortex well. Centrifuge at 14,000 rpm for 2 min and discard ethanol.
  9. The DNA pellet do not dry and dissolved immediately in 300 μl 1xTE, pH 8.0 at 55°C for 10-20 minutes.

Guanidine thiocyanate method for DNA extraction protocol

  • GuTC extraction buffer: 2 M guanidine thiocyanate, 10 mM Na3EDTA (pH ∼5.3);
    the final concentration of guanidine thiocyanate may need to optimized for certain plant/animals tissue from 0.5 to 4 M;
  • Chloroform-isoamyl alcohol mix (24:1);
  • 100% isopropanol (isopropyl alcohol, 2-propanol);
  • 70% ethanol;
  • Fresh 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0).
  1. 2 ml Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball freeze at -80°C, grind in the MM300 Mixer Mill for 2 min at 30 Hz.
  2. Add fresh 500 μl GuTC solution buffer (the sample volume should not exceed 20% of lysis buffer), vortex very well and incubate the samples at 60-65°C during 30-60 min.
  3. Add 1.5 volume (700 μl) of chloroform, vortex very well for 1 minute creating an emulsion. Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the upper aqueous layer to a new 2 ml microcentrifuge tube. Repeat this step.
  4. Transferred the upper aqueous layer to a new 2 ml microcentrifuge tube which contains of 800 μl 2-propanol, vortex well and centrifuge the tubes at maximum speed in a microcentrifuge for 3 minutes.
  5. Discard supernatant and wash pellet by adding 1.5 ml 70% EtOH, vortex well. Centrifuge at 14,000 rpm for 2 min and discard ethanol.
  6. The DNA pellet do not dry and dissolved immediately in 300 μl 1xTE, pH 8.0 at 55°C for 10-20 minutes.

CTAB method for DNA extraction protocol from herbarium

  • CTAB solution: 2% CTAB, 2 M NaCl, 10 mM Na3EDTA (pH ∼5.3);
  • Silica-membrane based spin columns (for example: QIAprep/QIAquick Spin Columns)
  • Chloroform-isoamyl alcohol mix (24:1);
  • 96% ethanol;
  • 80% ethanol, 10 mM Tris-HCl pH 7.5;
  • 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0).
  1. 2 ml Eppendorf Safe-Lock microcentrifuge tube with herbarium sample and glass ball (6 mm) grind in the MM300 Mixer Mill for 15 min at 30 Hz.
  2. Add fresh 350 μl CTAB solution buffer and add 700 μl of chloroform, vortex very well for creating an emulsion (in the MM300 Mixer Mill for 1 min at 30 Hz).
  3. Wait till the homogenate is allowed to separate into a clear upper aqueous layer and remove the lower phase (chloroform) contains dissolved polysaccharides.
  4. Add again 2 volume (700 μl) of chloroform, vortex very well (in the MM300 Mixer Mill for 1 min at 30 Hz); incubate the samples at 60-65°C during 30-60 min.
  5. Remove the lower phase (chloroform) and spin at maximum speed in a microcentrifuge for 5 minutes.
  6. Transferred the upper aqueous layer to a new 1.5 ml microcentrifuge tube which contains of 700 μl of 96% ethanol, vortex well. Do not centrifuge.
  7. Transfer up to 800 μl of the sample, to an spin column placed in a 2 ml collection tube. Close the lid, and centrifuge for 30 s at ≥8000 x g. Discard the flow-through.
  8. Wash the spin column by adding 700 μl 80% ethanol and centrifuging for 60 s.
  9. Discard the flow-through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer.
  10. Place the spin column in a new 1.5 ml collection tube. Add 200 μl 1xTE directly to the spin column membrane. Incubate the samples at 55°C during 10 min.
  11. Centrifuge for 1 min at ≥8000 x g to elute the DNA.

Proteinase K method for DNA extraction protocol

  1. In 2 ml Eppendorf Safe-Lock tube with mechanically disrupted animal or plant tissues add fresh 500 μl of extraction buffer (0.8 M guanidine thiocyanate, 10 mM EDTA, 5% Tween 20, 0.5% Triton X-100, 50 mM HEPES, pH 5.3*) with 200 μg of proteinase K, vortex very well and incubate the samples at 55°C for several hours or better overnight at 37°-55°C (the longer the better, until dissolve tissue) with occasional vortexing.
  2. Add 700 μl of chloroform, vortex very well for 1 minute creating an emulsion (in the MM300 Mixer Mill at 30 Hz); optionally: incubate the samples at 55°C during 30 min.
  3. Spin at maximum speed in a microcentrifuge for 5 minutes.
  4. Transfer the supernatant into a new 2 ml tube containing 500 μl of 2-propanol and 100 μl 3M Na-acetate, vortex very well, and centrifuge the tubes at maximum speed in a microcentrifuge for 4 minutes.
  5. Discard the supernatant and add 1.8 ml of 70% ethanol into tube and vortex well; centrifuge the tube for 5 minutes at 14000 rpm and again discard the supernatant.
  6. Do not dry DNA pellet and dissolved immediately in 300 μl of 1xTE, pH 8.0 (with RNAse A) at 55°C for 10-20 minutes.

* alternative extraction buffers for proteinase K:

  • 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0;
  • 3 M guanidine hydrochloride, 10 mM EDTA, 5% Tween 20, 0.5% Triton X-100, 30 mM Tris-HCl, pH 8.0
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