Gel electrophoresis
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GEL ELECTROPHORESIS. Gel Electrophoresis. Separation of molecules by size, charge, or shape PAGE ( P oly A crylamide E el E lectrophoresis ) Used to separate proteins Proteins are kept the same shape by detergent (SDS) Agarose Used to separate DNA fragments DNA can be recovered from gel.

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GEL ELECTROPHORESIS

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Gel electrophoresis

GEL ELECTROPHORESIS


Gel electrophoresis1

Gel Electrophoresis

  • Separation of molecules by size, charge, or shape

  • PAGE (PolyAcrylamideEel Electrophoresis)

    • Used to separate proteins

    • Proteins are kept the same shape by detergent (SDS)

  • Agarose

    • Used to separate DNA fragments

    • DNA can be recovered from gel


Agarose gel

Agarose Gel

  • Linked dextrose sugars leave channels for the DNA segment to “snake through”

  • Larger the fragment, the longer it takes for the DNA to travel

  • Gel is placed in a conductive buffer

  • Chamber delivers electricity

  • DNA distance can be gauged by watching loading dye


Dna gels

DNA is negative (PO4 backbone)

It runs to the positive electrode

The further it moves, the shorter the DNA

DNA Gels


How can you adjust ge

How can you adjust GE?

  • Run gels faster

    • Higher voltage

    • Lower temp to keep gels from melting

    • Does not affect resolution

  • Make agarose more concentrated

    • See smaller differences

    • More concentrated = more separation


Gel electrophoresis

Gels

  • This is an actual restriction digest

  • You can measure the distance the DNA traveled from the gel

  • Then you can determine the size of unknown fragments as long as you have a known


Size determination

Size Determination

  • Graph the known fragment sizes on semi-log paper

  • Find the distance the unknown traveled

  • Then extrapolate to the y axis to find the size


Lambda dna

Lambda DNA


Lambda dna digest

Lambda DNA digest


Lambda restriction sites

Lambda Restriction Sites

  • KNOWNS

  • HindIII:

    • 23,130

    • 9,416

    • 6,557

    • 4,361

    • 2,322

    • 2,027

  • UNKNOWNS

  • BamHI:

    • 16,841

    • 7,233

    • 6,770

    • 6,527

    • 5,656

  • EcoRI:

    • 21,226

    • 7,421

    • 5,804

    • 5,643

    • 4,878

    • 3,530


Gel electrophoresis

RFLP

  • Restriction fragment length polymorphism

  • Differences in DNA sequence on homologous chromosomes that can result in different patterns of restriction fragment lengths

    • Based on differences in alleles

    • On average, 2 humans differ 1 in 1000bp

      • These 2 people will differ in 3 million bp

  • Rarely have same exact distance between these restriction sites = polymorphic


Southern blotting

Southern Blotting

  • Used to verify the presence or absence of a specific nucleotide sequence in different DNA samples


Southern blotting1

Southern Blotting


Dna fingerprinting

DNA Fingerprinting

  • Different peoples’ DNA have different restriction sites

  • Restriction digests on our DNA cut different places, giving unique banding patterns

  • This is your DNA fingerprint


Dna fingerprinting1

DNA Fingerprinting

  • CSI

  • Match crime scene RFLPs to suspect RFLPs

    • The match is the killer

  • Paternity testing


Figure 20 17 dna fingerprints from a murder case

Figure 20.17 DNA fingerprints from a murder case


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