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 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, 10 mM Na3EDTA, 0.1 M MOPS, pH ∼4.6; the final concentration of guanidine thiocyanate may need to optimized for certain plant 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 plants with high concentration of pectin;

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Chloroform-isoamyl alcohol mix (24:1); 100% isopropanol (isopropyl alcohol, 2-propanol); 70% ethanol; 10 M lithium chloride; Fresh Milli-Q water (or Milli-Q ultrapure BioPak water) or 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0). When an ultrafiltration cartridge (BioPak) is utilized at the point-of-use, the water is suitable for genomics applications and cell culture.

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 and add 0.5 ml of chloroform, vortex very well (in the MM300 Mixer Mill for 3 min at 30 Hz) incubate the samples at 60°C for 30 minutes.

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 0.6 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 2 minutes. 6. Repeat the DNA extraction with chloroform until there will be no interphase. 7. Transferred the upper aqueous layer to a new 2 ml microcentrifuge tube which contains of 0.8 ml 2-propanol, mix well and immediately centrifuge at 14,000 rpm for 2 min. 8. 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. 9. 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. SDS method for DNA extraction, protocol 1. 2 ml Eppendorf Safe-Lock 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 Eppendorf Safe-Lock tube with mechanically disrupted seeds or leaves or herbarium add fresh 1 ml of extraction buffer (1% SDS, 3 mM CaCl2, 10 mM EDTA, 0.1 M HEPES, pH 5.3) with 200 μg of proteinase K, vortex very well and incubate the samples at 60°C for 30-60 minutes. 3. An additional isolation step may be required for samples with high content of polysaccharides or extracellular material, remove insoluble material from the extract by centrifugation at maximum speed on table microcentrifuge for 5 minutes. Transfer the cleared solution to a fresh tube. 4. Add 0.3 ml 3 M K-acetate (pH 4.8) to the lysate, vortex very well, and incubate for 5 min on ice; centrifuge the tube for 10 minutes at 14000 rpm, 4°C. 5. Optional: transferred the upper aqueous layer to a new 2 ml microcentrifuge tube which contains 0.6 ml of phenol-chloroform and vortex well (30 sec with max speed). Spin at maximum speed in a microcentrifuge for 3 minutes, RT. 6. Transfer the supernatant into a new 2 ml tube containing 0.5 ml of chlorophorm and vortex very well (30 sec at maximal speed). Centrifuge the tubes for 2 minutes at 14000 rpm. 7. Repeat the DNA extraction with chloroform until there will be no interphase. 8. Transfer the supernatant into a new 2 ml tube containing 0.8 ml of 2-propanol, mix very well, and immediately centrifuge the tubes for 2 minutes at 14000 rpm, RT. 9. Discard the supernatant and add 1.5 ml of 70% ethanol into tube and vortex well; centrifuge the tube for 2 minutes at 14000 rpm and again discard the supernatant. 10. 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. Express method for DNA extraction (direct PCR), protocol 1. In 1.5 ml Eppendorf Safe-Lock tube with mechanically disrupted leaves (100200 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 60°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.

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