Separating Science; One Band At A Time!

What is gel electrophoresis?

Gel electrophoresis is a method used in the lab that can separate RNA, DNA or proteins from one another. The molecules of interest are pushed through a porous gel by an electric current. One end of the gel has a positive charge, while the other end is negatively charged. For negatively charged molecules like DNA and RNA, they will end up towards the positively charged side of the gel. For proteins, they have many different charges so they have to be neutralized first. We neutralize them using sodium dodecyl sulfate (SDS), which ensures the separation on the gel is due to only size. The pores in the gel are small, so larger molecules don't travel as far as smaller molecules. The results from gel electrophoresis are bands in differing locations on the gel. How to interpret results is dependent on your molecule of interest.

What do we need to run gel electrophoresis?

• Gel box

• Gel with wells created at the negatively charged end (for sample placement)

• Running buffer (used to conduct charge over the gel)

• Power supply (creates electric current from each end)

• Visualization system (UV light to view DNA/RNA)

Gel Electrophoresis Steps:

1. Prepare samples: Add loading buffer to your sample

   1. loading buffer: contains glycerol (to make your sample more dense) and a dye (to make your sample visible while you place it in the wells)

2. Prepare gel and buffer: Buy/make gel and buffer suited for the type of molecule you're separating (ex: TAE or TBE buffer and agarose gel for DNA separation)

   1. If making gel in the lab, you must melt it down and set a separating comb in it while it cools (creates wells of certain size and abundance in the gel)

   2. Substances such as Ethidium Bromide can be added to the gel so we can later visualize the bands under UV light ( wear proper PPE when using this chemical!)

3. Load samples: Use a known 'ladder' sample to compare your samples later.

   1. Use a pipette to load your samples into each well. Make sure to label where each sample is in the gel!

4. Run the gel: Once the samples are loading, close the lid and plug the cords into the power supply

   1. run-time is dependent on the certain experiment you are doing (45-90 minutes common).

5. Visualize bands: For DNA and RNA, the bands can be visualized under UV light if the fluorescent dye has been added prior to running the gel. Proteins may need additional steps to view the bands (western blot).

   1. band locations are compared to detect presence of certain fragments, and/or size of fragments