Gel electrophoresis is a widely used laboratory technique that separates molecules, such as DNA, RNA, or proteins, based on their size and electrical charge. This method uses an electric field to move charged molecules through a gel matrix. Smaller molecules move more quickly and further through the gel, while larger molecules are hindered and travel shorter distances. The fundamental purpose of this separation is to analyze and purify biological molecules for various research and diagnostic applications.
Essential Components
Setting up a gel electrophoresis system requires equipment and reagents. The chamber holds the gel and running buffer. A power supply connects to the chamber, providing the electrical current necessary to drive molecules through the gel. A casting tray and comb form the gel with wells.
Buffers include a running buffer, which fills the chamber to conduct electricity, and a loading buffer, mixed with samples. The gel material is agarose powder for DNA and RNA separation, or polyacrylamide for proteins. A DNA ladder or protein marker serves as a reference to determine the size of molecules.
Preparing the Gel
Preparing the gel is a step in the electrophoresis process. Agarose gel is used, with concentrations ranging from 0.7% to 2.0% (w/v). A 1% agarose gel is common for general separation. Prepare by weighing agarose powder and mixing with running buffer (e.g., Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE)).
Heat this mixture until the agarose dissolves and becomes clear, avoiding boiling over. After the solution cools, pour it into a gel casting tray with taped ends. Carefully insert a comb into the still-liquid agarose. Allow the gel to solidify, which takes 20 to 45 minutes depending on size and concentration.
Assembling the Electrophoresis System
Once solidified, assemble the system. Gently remove the comb from the gel. Place the gel (in its tray or transferred) into the chamber. Orient the gel so sample wells are at the negative electrode (cathode) end, especially when separating negatively charged molecules like DNA.
Pour running buffer into the chamber until it covers the gel and fills the wells. Sufficient buffer volume ensures electrical flow and prevents gel drying. Secure the chamber lid and connect the leads to the power supply, ensuring positive (red) to anode and negative (black) to cathode.
Loading Samples and Initiating the Run
Before starting the run, prepare and load samples. Each sample, with a DNA ladder or protein marker, is mixed with loading dye. Loading dye adds density, allowing samples to sink into wells, and contains tracking dyes to indicate run progress. These tracking dyes do not bind to nucleic acids or proteins but move at a predictable rate relative to them.
Load each sample into a well using a micropipette. Precision is important to avoid cross-contamination or puncturing the well. Once loaded, turn on the power supply and set the appropriate voltage or current. For DNA agarose gels, common voltage settings range from 80 to 150 volts, depending on gel size and desired run time. As current flows, small bubbles form at the electrodes, confirming the electrical circuit is complete.
Addressing Setup Challenges
During gel electrophoresis setup, common issues arise. Gel failing to solidify often occurs if the agarose solution was not heated enough or poured before cooling enough. Reheating and ensuring dissolution, then allowing cooling before pouring, can resolve this. Air bubbles within the gel distort sample migration paths. Pouring the gel slowly and allowing it to settle before inserting the comb can minimize bubble formation.
Wells collapsing hinder sample loading. This usually happens if the comb is removed too quickly or if the gel is not solidified. Ensuring the gel is firm before comb removal and lifting the comb straight up prevents this.
Buffer leakage from the casting tray before the gel solidifies indicates improper sealing, requiring better sealing. Incorrect electrode connection between the chamber and power supply is fixed by checking the red-to-red and black-to-black connections. Difficulty loading samples can be due to a clogged pipette tip or trying to load too quickly; using a fresh tip and slow, steady pipetting helps.
References
Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press.
Green, M. R., & Sambrook, J. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor Laboratory Press.
Promega Corporation. (n.d.). Agarose Gel Electrophoresis. Retrieved from [https://www.promega.com/resources/pubhub/tpub_05/agarose-gel-electrophoresis/](https://www.promega.com/resources/pubhub/tpub_05/agarose-gel-electrophoresis/)
Bio-Rad Laboratories. (n.d.). Electrophoresis. Retrieved from [https://www.bio-rad.com/en-us/applications-technologies/electrophoresis](https://www.bio-rad.com/en-us/applications-technologies/electrophoresis)