Question

How to extract total dna

Answer 1

Extracting total DNA from a sample can be done using various methods, depending on the source of the DNA (e.g., animal, plant, bacterial cells) and the specific requirements for your experiment. Here is a general protocol that can be adapted for many types of samples:

### Basic Steps for DNA Extraction

#### Materials Needed:
- Sample (cells or tissue)
- Lysis buffer (detergent solution that breaks down cell membranes)
- Proteinase K (optional, for digesting proteins)
- RNase (optional, to remove RNA)
- Precipitation solution (usually alcohol, like isopropanol or ethanol)
- Buffer (like TE buffer or Tris-EDTA for re-suspending DNA)
- Centrifuge
- Pipettes and tips
- Tubes (microcentrifuge tubes)
- Water bath or incubator (set to 55°C, if using Proteinase K)

#### General Protocol:
1. **Sample Preparation:**
- Collect your sample (such as a small piece of tissue, blood, or cultured cells) and place it in a microcentrifuge tube.

2. **Cell Lysis:**
- Add a lysis buffer to the sample. This often contains detergents to disrupt cell membranes. You may include proteinase K if you want to digest proteins.
- Vortex to mix and then incubate at an appropriate temperature (e.g., 55°C for 1 hour) to allow for lysis.

3. **Removing Contaminants:**
- If using RNAase, add it to the mixture after lysis to remove RNA contaminants. Incubate for an additional 15-30 minutes at room temperature.

4. **DNA Precipitation:**
- Add an equal volume of cold isopropanol or ethanol to the lysis solution. This step causes DNA to precipitate out of the solution.
- Mix gently and then centrifuge at high speed (e.g., 12,000-15,000 rpm for 10-15 minutes) to pellet the DNA.

5. **Washing the DNA:**
- Carefully remove the supernatant, being careful not to disturb the DNA pellet.
- Wash the pellet with 70% ethanol (ethanol diluted with water) to remove any remaining impurities. Centrifuge again, then remove the ethanol.

6. **Resuspending the DNA:**
- Allow the pellet to air-dry for a few minutes, then resuspend the DNA in an appropriate buffer (like TE buffer) or sterile distilled water.

7. **Quantification:**
- Measure the concentration and purity of your extracted DNA using spectrophotometry (e.g., A260/A280 ratio).

### Notes:
- Always follow safety and laboratory protocols when handling biological materials and chemicals.
- The extraction can vary significantly based on the sample type, so it might be useful to search for protocols specific to your sample.
- Commercial DNA extraction kits are also available and can simplify the process, often providing optimized buffers and protocols to ensure high yield and purity.

This general guideline should help you extract DNA from various sources. Always consider the specific requirements and characteristics of your sample for the best results.