Gel Electrophoresis

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Electrophoresis  Used to separate and/or purify macromolecules  Commonly used for protein and nucleic acid separations  Separates molecules base on size, charge, or conformation using an electric field

Gel Electrophoresis  Uses a gel matrix for physical separation of molecules of interest  Gel is poured into a thin slab with wells to load sample into  The gel is immersed in a buffer containing ions to carry the current necessary for molecules to be moved through the gel and a buffering system to maintain pH

Agarose Gel  Agarose is derived from agar, a polysaccharide found in seaweed  Low concentrations of agarose solution can be heated in the appropriate buffer and form a solid gel when cooled  The gelling process can be reversed and solutions can be reheated and recooled if necessary  Solidified gels are flexible and stable, so they can be manipulated to study DNA

Agarose Gel  Often used in concentrations of 0.5% to 2%, with higher concentrations being “stiffer”  Fragments of various sizes can be separated using agarose, but resolution can be poor  Commonly used to analyze fragments of DNA

Agarose Gel  DNA has a net negative charge and migrates toward the positive pole  Smaller fragments move farther and faster through the gel, and larger fragments get “hung up” in the gel pores  The DNA moves through the gel at a rate roughly related to the inverse log of the number of base pairs present in the fragment

Agarose Gel  Bands of DNA must be stained in order to be seen  Ethidium bromide is commonly used, but requires UV light to visualize, and it is a known mutagen  Methylene blue stain may also be used to visualize bands of DNA and requires only visible light to see

Preparing a Gel  Determine the concentration of gel that is needed for the type of work being done  Obtain gel casting trays, gel boxes, and power supply  Sample combs are needed to make wells for loading DNA samples  Electrophoresis buffer is needed to maintain pH and conduct current  Loading buffer is needed to “weigh down” the sample and allow it to be tracked as it moves in the gel  A staining compound will need to be added to the sample or after electrophoresis in order to visualize the DNA  A light source will be needed to visualize bands of DNA

Pouring a Gel  Agarose solution of the desired concentration must be microwaved to completely melt all the agarose  The solution is allowed to cool to around 60C  The gel is poured into prepared casting trays with combs inserted  The gel is allowed to solidify at room temperature

Loading a Gel  After cooling at room temperature, the sample comb is carefully removed from the gel  The gel is loaded into the electrophoresis chamber and covered with buffer  DNA samples mixed with loading buffer are pipetted into the wells of the gel

Running a Gel  The electrodes are applied to the chamber and the appropriate current is turned on  The gel is allowed to electrophorese for the desired amount of time  At the end of the run, the gel is stained and destained for viewing (if not using EtBr)