PrimerDigital

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 or MOPS free acids) 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.

Materials for total DNA isolation

• CTAB solution: 1-2% CTAB, 2 M NaCl, 10 mM Na₃EDTA, 0.1 M MOPS (HEPES), pH ∼4.6;
  100 ml: 1-2 g CTAB, 2.1 g MOPS-acid, 2 ml 0.5 M Na₃EDTA, 40 ml 5 M NaCl;


• GuTC extraction buffer: 1 M guanidine thiocyanate, 1% N-lauroylsarcosine (Na salt, Sarkosyl), 10 mM Na₃EDTA, 0.1 M MOPS, pH ∼4.6;
  the final concentration of guanidine thiocyanate may need to optimized for certain plant/animals tissue from 0.5-1-2 M, but high concentration of guanidine thiocyanate (>1 M) may negatively interfere with the provision of high quality DNA for tissues with high concentration of pectin or polysaccharides (recommend using a concentration of 0.5 M of guanidine thiocyanate for tissues with a high concentration of polysaccharides, both in plants and animals);


• Chloroform-isoamyl alcohol mix (24:1);
• 100% isopropanol (isopropyl alcohol, 2-propanol);
• 70% ethanol;
• 10 M lithium chloride;
• Fresh 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0) or 1xTHE (1 mM EDTA, 5 mM Tris, 5 mM HEPES, pH 8.0).

CTAB or (Guanidine thiocyanate) method for DNA extraction protocol

1. 2 ml Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass boll freeze at -80°C, grind in the MM300 Mixer Mill for 3 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 1 ml CTAB or GuTC solution buffer (the sample volume should not exceed 20% of lysis buffer), vortex very well; optional: add 0.5 ml of chloroform, vortex very well (in the MM300 Mixer Mill for 3 min at 30 Hz); incubate the samples at 65°C within an hour.
3. Spin at maximum speed in a microcentrifuge for 2 minutes, transferred the upper aqueous layer to a new 2 ml microcentrifuge tube.
4. Add 0.5 ml of chloroform, vortex very well for 2 minute creating an emulsion (in the MM300 Mixer Mill at 30 Hz).
5. Spin at maximum speed in a microcentrifuge for 5 minutes.
6. Transferred the upper aqueous layer to a new 2 ml microcentrifuge tube which contains of 0.8 ml 2-propanol, vortex well and centrifuge the tubes at maximum speed in a microcentrifuge for 3 minutes.
7. Discard supernatant and wash pellet by adding 1.8 ml 70% EtOH, vortex well. Centrifuge at 14,000 rpm for 2 min and discard ethanol.
8. The DNA/RNA pellet do not dry and dissolved immediately in 300 μl 1xTE, pH 8.0 (with RNAse A) at 55°C for 10-20 minutes.

Proteinase K method for DNA extraction protocol

1. In 2 ml Eppendorf Safe-Lock tube with mechanically disrupted animal or plant tissues (seeds, leaves or herbarium) add fresh 1 ml of extraction buffer (0.8 M guanidine hydrochloride, 5 mM CaCl₂, 20 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 37°-55-65°C for several hours or better overnight at 37-55°C (the longer the better, until dissolve tissue) with occasional vortexing.
2. Add 0.7 ml of chloroform, vortex very well for 2 minute creating an emulsion (in the MM300 Mixer Mill at 30 Hz) and incubate the samples at 65°C for one hour.
3. Spin at maximum speed in a microcentrifuge for 5 minutes.
4. Transfer the supernatant into a new 2 ml tube containing 0.8 ml 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 1.7 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, 5 mM CaCl₂, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0;

0.8 M guanidine thiocyanate, 5 mM CaCl₂, 20 mM EDTA, 5% Tween 20, 0.5% Triton X-100, 30 mM Tris-HCl, pH 8.0

Express method for DNA extraction (direct PCR), protocol

1. In 1.5 ml Eppendorf Safe-Lock tube with mechanically disrupted leaves (100-200 μl total volume) with fresh 1ml extraction buffer (0.1 M NaOH, 0.1 M NaCl, 20 mM EDTA), vortex very well. Incubate the samples at 55°C for 10 minutes.
2. Centrifuge the tube for 10 minutes at 14000 rpm.
3. Transfer 100 μl of the supernatant into a new 1.5 ml tube containing 1 ml of 10 mM MOPS (free acid), pH 4.6 and mix well.
4. Use 0.5-1 μl of DNA solution directly to PCR.