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Gel Electrophoresis

Gel Electrophoresis• Gel electrophoresis

is used in order toseparate, identify,and purify 0.5 to25Kb DNA fragments.

• DNA has negativelycharged phosphatesalong the DNAbackbone.

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Gel Electrophoresis

• DNA fragments can beseparated by size whenapplied to an electric field.

• DNA molecules migratetoward the anode (+).

Gel Electrophoresis

• Agarose is a porous gelatinouscarbohydrate.

• The DNA samples are loadedinto an agarose gel mold.

• The agarose mold is placed intoa tank which contains a buffersolution (TAE or TBE)

• The gel is run at a voltage andfor a time period that willseparate the DNA fragments

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Gel electrphoresis

Gel Electrophoresis• Large fragments of DNA

move slowly through theagarose while small DNAfragments move quickly.

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Gel Electrophoresis• Low agarose concentrations.

– 0.3 to 0.5%– Separate large DNA fragments.

• 20 to 60kb

• Medium agarose concentrations.– 0.5% to 1.0%– Separate medium sized fragments.

• 0.5 to 30kb

• High agarose concentrations.– 1 to 1.5%– Separate small DNA fragments.

• 0.2 to 0.5kb

Gel Electrophoresis

• Molecular weight marker orladder - DNA fragments ofknown sizes.

• Loaded into a separate lane.• Sizes of unknown DNA

fragments can be estimatedusing a molecular weightmarker.

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Gel Electrophoresis• DNA cannot be seen during gel

electrophoresis.• Tracking Dye/Sample

Buffer/Loading Dye - used inorder to estimate how far theDNA has run during a gelelectrophoresis.

• Negatively charged.• Added to each DNA sample.• Does not bind to the DNA!

Gel Electrophoresis• Bromphenol Blue.

– Migrates with DNAfragments around 0.5kb.

• Xylene Cyanol– Xylene Cyanol migrates

with DNA fragmentsaround 5kb.

• Glycerol - Increases thedensity of the sample foreasier loading.

BMB

Xylene

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Gel Electrophoresis• Between 5 and 200 ng of a

single DNA fragment shouldbe loaded into a well.

• Question Are weoverloading our gels?– We digested 10ul of DNA at

about .1ug/ul.– How many ug did we digest?– The entire amount of each

digested sample will be addedto each well.

– >200ng of DNA is consideredoverloading.

Notoverloaded Overloaded

Gel Electrophoresis

• Ethidium Bromide(EtBr) - Used in order tovisualize the DNA.

• Intercalates (inserts)between DNA’s basepairs

• Fluoresces under UVlight

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Gel Electrophoresis• Longer DNA fragments will

contain more ethidiumbromide and appear darker

• Shorter DNA fragments willcontain less ethidiumbromide and appear lighter

Gel Electrophoresis• Ethidium bromide causes

nicks in the DNA fragmentsin response to UV light.– The nicking of the DNA will

cause the DNA fragments tomigrate slower.

• Ethidium bromide is amutagen.

• Wear gloves whenhandling anything whichcontains or containedethidium bromide.