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Total DNA isolation protocol

The procedure is suitable for all types of tissues from a wide variety of animal, blood, plant species and soil.
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 protocol for the isolation of DNA

  • CTAB solution: 2% CTAB, 1.5 M NaCl, 10 mM Na3EDTA, 0.1 M HEPES-acid;
    100 ml: 3 g CTAB, 2.4 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);
  • 70% ethanol;
  • 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0);
  1. 2 ml Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball (6 mm), grind in the MM300 Mixer Mill for 2-10 min at 30 Hz.
  2. In 2 ml tube with mechanically disrupted seeds/leaves/herbarium or DNA solution (CTAB purification) add 1 ml CTAB solution buffer, mix in the MM300 Mixer Mill for 2 min at 30 Hz and incubate the samples at 65°C during 1-2 or more hours.
  3. Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the clarified solution to a new 2 ml microcentrifuge tube contains an equal volume of chloroform.
  4. Mix very well for 3 minutes in the MM300 Mixer Mill at 30 Hz.
  5. Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the upper aqueous layer to a new 2 ml microcentrifuge tube which contains 600 μl 2-propanol, vortex well and centrifuge the tubes at maximum speed in a microcentrifuge for 2 minutes.
  6. Discard supernatant and wash pellet by adding 1.8 ml 70% EtOH, vortex very well. Centrifuge at maximum speed for 2 min and discard ethanol.
  7. The DNA pellet do not dry and dissolved immediately in 300 μl 1xTE, pH 8.0 at 55-60°C for 5-10 minutes.

CTAB protocol for the isolation of DNA from difficult tissues (high levels of secondary metabolites or polysaccharides), herbarium and soil

  • CTAB solution: 3% CTAB, 1.5 M NaCl, 10 mM Na3EDTA, 0.1 M MOPS-acid;
    100 ml: 3 g CTAB, 2.1 g MOPS-acid, 2 ml 0.5 M Na3EDTA, 30 ml 5 M NaCl;
  • Mini spin column: HiBind® (Omega Bio-tek) or NucleoSpin® (MN) or compatible with the following Qiagen kits: Qiaprep Spin Mini, QiaQuick Gel Extraction, QiaQuick PCR Clean-up, DNeasy Plant DNA;
  • Buffer QG (5.5 M guanidine thiocyanate, 20 mM Tris-HCl pH 6.6);
  • DNA Wash Buffer PE (80% ethanol, 20 mM NaCl, 2 mM Tris-HCl, pH 7.5);
  • 96% ethanol;
  • Elution buffer (0.5 mM EDTA, 10 mM Tris-HCl, pH 9.0);
  1. 2 ml Eppendorf Safe-Lock microcentrifuge tube with herbarium sample (the sample mass should not exceed 50 mg) and glass ball (6 mm) grind in the MM300 Mixer Mill for 15 min at 30 Hz.
  2. Add 1 ml CTAB solution buffer, vortex to mix thoroughly and incubate the samples at 65°C during 1-3 or more hours.
  3. Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the clarified solution to a new 2 ml microcentrifuge tube contain an equal volume of 96% ethanol (50-100% of the CTAB solution volume). Vortex to mix thoroughly.
  4. Insert a HiBind DNA Mini column into a 2 ml collection tube. Transfer up to 700 μl sample from step 3 to the HiBind DNA Mini column. Centrifuge at maximum speed for 30 sec at room temperature.
  5. Using the same collection tube, repeat previous step until all the CTAB mix solution has passed through the HiBind DNA Mini column. Centrifuge at maximum speed for 30 sec. Discard the filtrate and collection tube.
  6. Add 500 μl 96% ethanol. Centrifuge at maximum speed for 30 sec. Discard the filtrate and reuse collection tube.
  7. Add 500 μl QG buffer. Centrifuge at maximum speed for 30 sec. Discard the filtrate and reuse collection tube.
  8. Add 700 μl DNA wash buffer PE. Centrifuge at maximum speed for 30 sec. Discard the filtrate and reuse collection tube.
  9. Using the same collection tube, repeat previous step for second DNA wash buffer wash step. Centrifuge at maximum speed for 1 minute.
  10. Transfer the HiBind DNA Mini column into a clean 1.5 ml tube. Add 200 μl elution buffer and heat at 55°C for 10 minutes.
  11. Centrifuge at maximum speed for 1 minute. Store eluted DNA at -20°C.


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-acid*) 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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