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    Agarose Gel Electrophoresis

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    JL Deomampo
    Agarose gel electrophoresis is a technique used to separate DNA molecules by size. DNA samples are loaded into wells in an agarose gel and an electric current is applied, causing the negatively charged DNA to migrate through the gel at rates corresponding to their sizes. Smaller DNA fragments migrate farther than larger ones. After electrophoresis, DNA bands can be visualized by staining with ethidium bromide and exposing to UV light. A DNA ladder standard is run alongside samples to determine the sizes of unknown DNA fragments.

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    Agarose Gel Electrophoresis

    Uploaded by

    JL Deomampo
    1K views20 pages
    Agarose gel electrophoresis is a technique used to separate DNA molecules by size. DNA samples are loaded into wells in an agarose gel and an electric current is applied, causing the negatively charged DNA to migrate through the gel at rates corresponding to their sizes. Smaller DNA fragments migrate farther than larger ones. After electrophoresis, DNA bands can be visualized by staining with ethidium bromide and exposing to UV light. A DNA ladder standard is run alongside samples to determine the sizes of unknown DNA fragments.

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    Agarose Gel Electrophoresis.ppt

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    Agarose gel electrophoresis is a technique used to separate DNA molecules by size. DNA samples are loaded into wells in an agarose gel and an electric current is applied, causing the negatively charged DNA to migrate through the gel at rates corresponding to their sizes. Smaller DNA fragments migrate farther than larger ones. After electrophoresis, DNA bands can be visualized by staining with ethidium bromide and exposing to UV light. A DNA ladder standard is run alongside samples to determine the sizes of unknown DNA fragments.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    1K views20 pages

    Agarose Gel Electrophoresis

    Uploaded by

    JL Deomampo
    Agarose gel electrophoresis is a technique used to separate DNA molecules by size. DNA samples are loaded into wells in an agarose gel and an electric current is applied, causing the negatively charged DNA to migrate through the gel at rates corresponding to their sizes. Smaller DNA fragments migrate farther than larger ones. After electrophoresis, DNA bands can be visualized by staining with ethidium bromide and exposing to UV light. A DNA ladder standard is run alongside samples to determine the sizes of unknown DNA fragments.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
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    Agarose Gel Electrophoresis

    Agarose Gel Electrophoresis


    DNA (-) small large

    Gel electrophoresis
    +
    separates molecules different rates of movement through a gel under the influence of an electrical field( carrying within electricity) widely used technique for the analysis of:
    nucleic acids (Agarose Gel Electrophoresis) Proteins (SDS-PAGE)

    Power

    Agarose Gel
    Agarose Agarose is a linear polymer derived from red seaweed. Malasian word: agaragar. Pores = sieve Increasing agarose concentration:
    decreases pore size limits the size range of molecules that can be separated.
    D-galactose 3,6-anhydro L-galactose

    Scanning Electron Micrograph of Agarose Gel (11 m)

    1% agarose

    2% agarose

    DNA buffer wells

    Cathode (negative)

    Anode (positive)

    Sample Preparation and Loading


    6X Loading Buffer: Bromophenol Blue (tracking dye) Glycerol/ Glucose/ Sucrose (increase sample density)

    DNA Migration
    Size
    migration rate of DNA fragment and logarithm of its size (in basepairs): linear relationship (inverse) Larger molecules move more slowly because of more friction

    Electrical field strength Buffer (TAE, TBE) Agarose Gel Concentration Sample loading

    DNA Ladder Standard


    Serves as a marker to determine the sizes of unknown DNAs.
    12,000 bp 5,000

    DNA migration

    2,000 1,650 1,000 850 650 500 400 300 200 100

    bromophenol blue

    Visualization
    Ethidium bromide
    binds to DNA and fluoresces under UV light can be added to the gel and/or running buffer before electrophoresis or used as developing solution after electrophoresis CAUTION: Powerful mutagen and moderately toxic Decontamination
    Lunn and Sansone Method : + 20 mL 5% hydrophosphorous acid and 12 mL 0. 5 M sodium Nitrate for every 100 mL EtBr (20 hrs) Armour method: Bleach (2-3 days) Charcoal Filtration

    Safer Alternatives to Ethidium Bromide Methylene Blue BioRAD - Bio-Safe DNA Stain Wards - QUIKView DNA Stain Carolina BLU Stain
    advantages
    Inexpensive Less toxic No UV light required No hazardous waste disposal

    disadvantages
    Less sensitive More DNA needed on gel Longer staining/destaining time

    Visualizing the DNA (QuikVIEW stain)


    DNA ladder

    wells

    PCR Product

    + - - - - + + - - + - +

    2,000 bp 1,500 1,000 750 500 250

    Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia DNA

    Determining sample size

    DNA ladder

    DNA migration rate and logarithm of its size (in basepairs): inverse linear relationship

    base pairs

    Distance migrated

    DNA ladder

    x bp base pairs
    sample

    Distance migrated

    Results

    marker

    Grp. 1

    Grp. 2

    Grp. 3

    Grp. 4

    Grp. 5

    Grp. 6

    Grp. 7

    4 BIO 4
    RESULTS

    wells

    10,000 bp 8,000 6,000 5,000 4,000 3,000 2,500 2,000 1,500 1,000 750
    (From product insert of Promega 1 kb DNA ladder)

    4 BIO 3
    RESULTS
    Grp. 8 Grp. 7 Grp. 6 Grp. 5 Grp. 4 Grp. 3 Grp. 2 Grp. 1 marker

    wells

    (From product insert of Promega 1 kb DNA ladder)

    SAMPLE 3
    SAMPLE 2 SAMPLE 1 Grp. 7 Grp. 6 Grp. 5 Grp. 4 Grp. 3 Grp. 2 Grp. 1 marker

    4 BIO 5

    RESULTS

    wells

    (From product insert of Promega 1 kb DNA ladder)

    4 BIO 2
    RESULTS
    Grp. 7 Grp. 3 Grp. 8 Grp. 6 Grp. 5 Grp. 4 NONE Grp. 2 Grp. 1 marker

    wells

    (From product insert of Promega 1 kb DNA ladder)

    marker

    Grp. 1

    Grp. 2

    Grp. 3

    Grp. 4

    Grp. 5

    Grp. 6

    Grp. 7

    4 BIO 6
    RESULTS

    wells

    10,000 bp 8,000 6,000 5,000 4,000 3,000 2,500 2,000 1,500 1,000 750
    (From product insert of Promega 1 kb DNA ladder)

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