Isolation of DNA from Tissues and Qualitative Analysis by Agarose Gel Electrophoresis

Aim

To isolate DNA from animal tissue and analyze the isolated DNA qualitatively by agarose gel electrophoresis.

Principle

DNA is present inside the nucleus of cells. During isolation, cells are lysed using detergent to release DNA. Proteins and other contaminants are removed, and DNA is precipitated using chilled alcohol.

The isolated DNA is then analyzed by agarose gel electrophoresis. DNA molecules migrate through agarose gel under an electric field towards the positive electrode because DNA is negatively charged due to phosphate groups.

The movement of DNA can be represented as:

$\text{DNA}^{-} \rightarrow \text{Positive Electrode (Anode)}$

Smaller DNA fragments move faster through the gel compared to larger fragments.

Requirements

Apparatus

• Beaker
• Test tubes
• Micropipette
• Centrifuge
• Electrophoresis unit
• Power supply
• Gel casting tray
• UV transilluminator

Chemicals/Reagents

1. Tissue sample (liver/onion/animal tissue)
2. Extraction buffer
3. SDS detergent
4. Proteinase K (optional)
5. Chilled ethanol/isopropanol
6. Agarose
7. TAE/TBE buffer
8. Ethidium bromide or safe DNA stain
9. Loading dye
10. DNA marker/ladder

Procedure

Part A: Isolation of DNA

Step 1: Tissue Homogenization

1. Take a small amount of tissue sample.
2. Grind the tissue in extraction buffer using a mortar and pestle.

Step 2: Cell Lysis

1. Add SDS detergent to break the cell membrane and nuclear membrane.
2. Mix gently.

Step 3: Removal of Proteins

1. Add Proteinase K if available.
2. Centrifuge the mixture to remove cell debris and proteins.

Step 4: DNA Precipitation

1. Transfer the supernatant to another test tube.
2. Add chilled ethanol slowly along the side of the tube.
3. White thread-like DNA precipitate appears.

Step 5: Collection of DNA

1. Spool out DNA using a glass rod or pipette tip.
2. Transfer DNA into distilled water or buffer.

Part B: Agarose Gel Electrophoresis

Step 1: Preparation of Agarose Gel

1. Prepare 1% agarose in TAE/TBE buffer.
2. Heat until agarose dissolves completely.
3. Add DNA stain.
4. Pour into gel tray and insert comb.
5. Allow the gel to solidify.

Step 2: Loading of Samples

1. Remove comb carefully.
2. Place gel in electrophoresis chamber.
3. Add buffer to cover gel.
4. Mix DNA sample with loading dye.
5. Load DNA samples and DNA marker into wells.

Step 3: Electrophoresis

1. Connect electrodes properly.
2. Run electrophoresis at 80–100 V for about 30–45 minutes.

Step 4: Observation

1. Observe gel under UV transilluminator.
2. DNA bands appear as fluorescent bands.

Observation

During DNA Isolation

• White thread-like precipitate observed after adding chilled ethanol.

During Electrophoresis

• DNA bands observed in agarose gel under UV light.

Interpretation

1. Presence of distinct DNA bands indicates successful isolation of DNA.
2. Smearing may indicate degraded DNA.
3. Thick bands indicate high DNA concentration.