Lab 10: DNA Gel Electrophoresis

Describe how DNA agarose gel electrophoresis works

• Separate DNA molecules based on their size

• DNA sample is loaded on an agarose gel matrix

• Electric field is applied to the agarose once DNA is applied

• DNA is negatively charged due to its phosphate groups, so the DNA fragments will migrate through the gel matrix

  1. Depending on the size of the DNA molecules, the migration will happen at different rates

agarose gel

contains the DNA for electrophoresis

loading dye

allows for tracking the migration of DNA during electrophoresis and determines when the separation of the DNA is complete

running buffer

added to the PCR and a higher density than water, like glycerol or sucrose to sink the DNA mixture into the well

electric current

allows the movement of the DNA from the side near the negative electrode to the positive electrode

DNA staining dye

allows the dye to intercalate b/n DNA base pairs so that they can fluorescence under UV light

molecular marker (ladder)

1. a mixture of DNA products with known sizes

   1. Serves as a reference for estimating the sizes of the unknown DNA fragments

   2. Products in the ladder can be used to determine the sizes of the amplified DNA

UV light

shows where the DNA is located in the gel matrix

Describe how to load and run DNA samples on a gel.

1. The get matrix is prepared for DNA loading

   1. Agarose gel matrix is cast in a mold with a comb in order to create wells in it for the DNA to go in

2. The DNA samples are loaded into the wells

   1. Samples are mixed with loading buffer 

3. Apply the electric current

   1. Electrophoresis chamber has electrodes at either end of the gel, creating an electric field

   2. DNA will move towards the positive electrode, which is why they’re placed so close to the negative electrode

   3. Large pieces will move slower compared to the smaller pieces because the gel will act like a sieve 

4. Result analysis

   1. Can take between 30-60 minutes

   2. Gel is stained with a DNA-binding dye so that the dyes can intercalate b/n the DNA base pairs and fluorescence under UV light, signaling where the DNA products are located