Describe how you estimate DNA fragment size using gel electrophoresis?

To estimate DNA fragment size using gel electrophoresis, you first need to prepare a gel made of agarose or polyacrylamide. The gel is then placed in a buffer solution and a voltage is applied across the gel. DNA samples are loaded into wells at one end of the gel and the voltage causes the DNA fragments to migrate through the gel towards the positive electrode.

As the DNA fragments migrate through the gel, smaller fragments move faster and travel further than larger fragments. This separation is based on the size and shape of the DNA molecules. After the electrophoresis is complete, the gel is stained with a dye that binds to the DNA and makes the bands visible under UV light.

To estimate the size of the DNA fragments, a DNA ladder or molecular weight marker is run alongside the samples. The ladder contains DNA fragments of known sizes, allowing you to compare the migration distance of the unknown fragments to the ladder fragments. By measuring the distance traveled by the DNA fragments and comparing it to the ladder, you can estimate the size of the unknown fragments.

Gel Electrophoresis for DNA Fragment Size Estimation

Materials:

- Agarose gel
- DNA samples of known sizes
- DNA fragment samples
- Gel electrophoresis apparatus
- Visualizing device (e.g., UV transilluminator)
- Molecular weight marker

Procedure:

1. Prepare agarose gel: Dissolve agarose powder in buffer and heat to melt. Pour the melted agarose into a gel mold and let it solidify.

2. Load DNA samples: Mix each DNA sample with loading dye and load onto the gel. Include known size DNA fragments as standards.

3. Run electrophoresis: Connect the gel to an electrophoresis apparatus and apply a voltage. The DNA fragments will migrate through the gel, with smaller fragments moving faster than larger ones.

4. Visualize DNA fragments: After electrophoresis, stain the gel with a DNA stain (e.g., ethidium bromide) and visualize under a UV transilluminator.

5. Compare fragment sizes: Measure the distance each DNA fragment has traveled from the loading well.

Estimation using Molecular Weight Marker:

- Plot a calibration curve using the known size DNA fragments (molecular weight marker). The x-axis represents the fragment size (bp) and the y-axis represents the distance traveled.
- Locate the distance traveled by the unknown DNA fragment on the calibration curve.
- Read the fragment size corresponding to this distance from the x-axis.

Estimation without Molecular Weight Marker:

- Assume a linear relationship between fragment size and distance traveled.
- Measure the distances traveled by two or more known size fragments and plot the points on a graph.
- Extrapolate the line formed by the points to the distance traveled by the unknown fragment.
- Read the estimated fragment size from the x-axis.

Factors Affecting Fragment Size Estimation:

- Gel concentration: Higher agarose concentrations result in slower migration of DNA fragments.
- Electrophoresis time: Longer electrophoresis times allow for better separation of fragments.
- Voltage: Higher voltages can cause DNA fragments to run faster and potentially damage them.
- Gel temperature: Higher temperatures can affect the migration rate of DNA fragments.