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Gel Electrophoresis. Electrophoresis  Used to separate and/or purify macromolecules  Commonly used for protein and nucleic acid separations  Separates.

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Presentation on theme: "Gel Electrophoresis. Electrophoresis  Used to separate and/or purify macromolecules  Commonly used for protein and nucleic acid separations  Separates."— Presentation transcript:

1 Gel Electrophoresis

2 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

3 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

4 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

5 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

6 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

7 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

8 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

9 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

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11 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

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13 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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16 Disclaimer   This workforce solution was funded by a grant awarded under the President’s Community-Based Job Training Grants as implemented by the U.S. Department of Labor’s Employment and Training Administration. The solution was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor. The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership. This solution is copyrighted by the institution that created it. Internal use by an organization and/or personal use by an individual for non-commercial purposes is permissible. All other uses require the prior authorization of the copyright owner.


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