Universal DNA isolation protocol
Ruslan Kalendar
Abstract
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).
Steps
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.
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).
2h 0m 0s
65
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.
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.
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).
Centrifuge at maximum speed in a microcentrifuge for 2-5 minutes. A whitish DNA pellet should be visible.
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.
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.
Do not dry the DNA pellet and immediately dissolve it in 300 μl 1xTE, pH 8.0 at 55°C for 10-20 minutes.
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.
