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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.