Gel Electrophoresis

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)

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