TITLE: Universal DNA isolation protocol
AUTHORS: Ruslan  Kalendar

[DESCRIPTION]
The isolation of nucleic acids from a sample is an important step for many molecular biological applications and medical diagnostic assays. This protocol describes an efficient method for purification or/and isolation of nucleic acids from difficult animal tissues, plant material and other samples from which DNA extraction is generally regarded as being difficult which can contain impurities and inhibitors or interfering substances. This method is established acidic CTAB (with a pH value of 5 to 6.8) based extraction protocol that allows for reliable isolation of high molecular weight genomic DNA for removing contaminants from nucleic acids in a sample, e.g., environmental or biological samples such as soil, food, plant, animal or microorganism. DNA may quickly be extracted from samples without phenol, guanidine thiocyanate or 2-mercaptoethanol.

[BEFORE_START]
Required solutions:
CTAB solution: 1.5% CTAB, 1.5 M NaCl, 10 mM Na3EDTA, 0.1 M HEPES/MOPS (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.
Fresh 1xTE (1 mM EDTA, 10mM Tris-HCl, pH 8.0).
Ribonuclease A solution: 10 mg/ml in 50% glycerol, 10 mM Tris-HCL pH 8.0.
100% Isopropanol (2-propanol).

[GUIDELINES]
This protocol for DNA isolation is universal for most biological specimens. The method will effectively isolate DNA from whole blood, bones, plant samples, soil, herbarium, the mycelium of fungi, and tissues rich in secondary metabolites, polysaccharides, and pigments. DNA samples obtained using the proposed method can be used in studies where the presence of contaminants in nucleic acids is undesirable; for example, during cloning, sequencing, and genotyping.

All lab procedures are performed at room temperature. Prepare all solutions using ultrapure Milli-Q water and analytical grade reagents. Prepare and store all reagents at room temperature unless otherwise specified and away from direct sunlight. 

The composition of the lysis solution contains inorganic salts (sodium chloride), within the effective concentration in the range of 1–4 M. The optimal concentration of the detergent is 1.5% CTAB. To increase the efficiency of DNA extraction, proteinase K can be added to the acidic lysing solution, which retains proteolytic activity at high ionic strength and low pH values, even in the presence of strong detergents and chaotropic agents. The subsequent extraction with chloroform increases the purity of the isolated DNA, especially from complex samples (thermally treated raw materials, blood, herbarium specimen, and soils). The effective concentration of chloroform is 1–2 volumes of the total lysate. Further, the DNA is precipitated from the aqueous phase with a water-soluble organic solvent, such as ethanol (it is preferable to use isopropanol). Depending on the biological material, DNA can be precipitated by filtration through a column with a glass microfiber filter, for example, Glass microfiber filters (Grade GF/A) or through cellulose paper. Finally, DNA is washed by precipitation or filtration in a solution of 80% ethanol and dissolved in low ionic buffered water.

[STEPS]
1. Eppendorf Safe-Lock microcentrifuge tube with tissue sample and glass ball (6 mm) freezes at -80°C, grind in the MM300 Mixer Mill for 2 min at 30 Hz. Alternatively, grind the sample in the lysis solution, so that there is enough empty space in the tube.

2. In a 2 ml tube with mechanically disrupted tissue/seeds/leaves/herbarium or DNA solution (CTAB purification) add 1 ml CTAB solution buffer with RNAse A (the sample mass should not exceed 100 mg), vortex thoroughly, and incubate the samples at 55-65°C during 30-120 min or longer (long incubation increases DNA yield).
120 min
65 10
3. Optionally, spin at maximum speed in a microcentrifuge for 2 minutes, and preferably transfer the entire clarified supernatant to a new 2 ml Safe-Lock microcentrifuge tube.
12.  Transfer the entire clarified supernatant to a new 2 ml microcentrifuge tube contains an equal volume of chloroform. Mix well for 3-5 minutes in the MM300 Mixer Mill at 30 Hz.
4. Add to the tube an equal volume of chloroform. Mix well for 1-5 minutes in the MM300 Mixer Mill at 30 Hz or vortex thoroughly for 1 minute. Spin at maximum speed in a microcentrifuge for 1-3 minutes.
5. Transfer the entire clarified upper aqueous layer to a new 2 ml microcentrifuge tube which contains half to an equal volume of 2-propanol and vortex thoroughly (5-10 secs).
6. Centrifuge at maximum speed in a microcentrifuge for 2-5 minutes. A whitish DNA pellet should be visible.
7. Discard the supernatant and wash the pellet by adding 1.5-1.8 ml 70% ethanol, vortex thoroughly. At this stage, DNA samples can be stored at room temperature or refrigerated.
8. Centrifuge at maximum speed for 2 min and carefully discard the supernatant by decanting or with a micropipette. A whitish DNA pellet should be visible during discarding a supernatant.
9. Do not dry the DNA pellet and immediately dissolve it in 300 μl 1xTE, pH 8.0 at 55°C for 10-20 minutes.