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    Southern W04

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    Chalidin Sz
    Southern blotting is used to detect specific DNA sequences. It involves restriction digestion of DNA, gel electrophoresis to separate fragments by size, transfer to a membrane, and probing to detect the DNA. In this laboratory, students will perform restriction digestion on DNA samples, run the fragments on a gel, complete depurination and denaturation steps, and set up an overnight transfer of DNA from the gel to a nylon membrane. The next step will be to detect the DNA bands on the membrane using a non-radioactive probe and staining method.

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    Southern W04

    Uploaded by

    Chalidin Sz
    73 views15 pages
    Southern blotting is used to detect specific DNA sequences. It involves restriction digestion of DNA, gel electrophoresis to separate fragments by size, transfer to a membrane, and probing to detect the DNA. In this laboratory, students will perform restriction digestion on DNA samples, run the fragments on a gel, complete depurination and denaturation steps, and set up an overnight transfer of DNA from the gel to a nylon membrane. The next step will be to detect the DNA bands on the membrane using a non-radioactive probe and staining method.

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    Southern blotting is used to detect specific DNA sequences. It involves restriction digestion of DNA, gel electrophoresis to separate fragments by size, transfer to a membrane, and probing to detect the DNA. In this laboratory, students will perform restriction digestion on DNA samples, run the fragments on a gel, complete depurination and denaturation steps, and set up an overnight transfer of DNA from the gel to a nylon membrane. The next step will be to detect the DNA bands on the membrane using a non-radioactive probe and staining method.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    73 views15 pages

    Southern W04

    Uploaded by

    Chalidin Sz
    Southern blotting is used to detect specific DNA sequences. It involves restriction digestion of DNA, gel electrophoresis to separate fragments by size, transfer to a membrane, and probing to detect the DNA. In this laboratory, students will perform restriction digestion on DNA samples, run the fragments on a gel, complete depurination and denaturation steps, and set up an overnight transfer of DNA from the gel to a nylon membrane. The next step will be to detect the DNA bands on the membrane using a non-radioactive probe and staining method.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 15

    Southern Blotting

    General Scheme for Southern Blot

    Restriction Digest
    Gel Electrophoresis
    DNA Preparation:
    Denaturation/Depurination

    Transfer to filter: Blotting

    Detecting DNA: Probing


    Southern Blot Analysis
    Southern Blot
    • Restriction Digest of DNAdone

    • Electrophoresis
    • Denaturation/Depurination
    • Blotting Step (overnight)
    • Detection of DNA (next lab)
    Format for today’s laboratory:
    • 5 groups

    • Each group
    – loads and runs agarose gel
    – Completes depurination/denaturation steps
    – Sets up DNA transfer
    DNA Fingerprinting Analysis
    40 ul sample per well
    Sample Tube
    Label
    Standard DNA fragments A
    Mother DNA cut with Enzyme B
    Child DNA cut with Enzyme C
    Possible Father #1 DNA cut D
    with Enzyme
    Possible Father #2 DNA cut E
    with Enzyme
    Depurination and Denaturation Step
    page 3-83 with changes

    • Place gel in 100 ml 0.25N HCl for a maximum


    of 8 minutes.
    • Rinse gel several times with 100 ml distilled
    water
    • Place gel in 100 ml DNA denaturation solution
    for 15 min. with occasional shaking.
    • Replace with 100 ml fresh denaturation solution
    for 15 minutes
    The reason for depurination
    • Depurination: 0.25 N HCl
    (used to help in a teaching lab)

    – Reduces binding between DNA strands by


    creating apurinic sites (loss of purine base)
    – Predisposes the DNA-phosphate backbone
    to cleavage by alkaline solution
    – Smaller fragments transfer better
    The reason for denaturation:
    • Denaturation:NaOH/NaCl

    – Double-stranded DNA is converted to


    Single-stranded DNA
    – Sugar-phosphate backbone is cleaved at
    the sites of depurination
    – This step must always be performed.
    Setting up the Southern Blot Transfer
    page 3-84

    • Line a tray with plastic wrap

    • Place “denatured” gel upside down on wrap

    • Pre-wet nylon membrane in denaturation


    solution for 5 minutes

    • Place nylon on top of inverted gel


    Setting up the Southern Blot Transfer
    Page 3-84
    • Place filter paper on top of nylon membrane
    • Remove air bubbles
    • Place stack of paper towels on top of filter
    paper
    • Place empty 400 ml beaker on towel
    • Incubate overnight
    Setting up the Southern Blot Transfer
    Order of Components from the bottom:
    Gel
    Membrane
    Filter paper
    Paper towels
    Weight
     Overnight Incubation at Room Temperature
    Single stranded DNA will diffuse by capillary
    transfer from gel to nylon membrane
    Analysis of Southern Blot

    A Non-isotopic Method of DNA Detection


    Revised from Lab Manual Instructions
    Comparing Our Experimental System
    to Southern Blots in Research Labs
    In research lab In teaching lab
    Radioisotopic Method Non-Isotopic method
    DNA Restriction Restriction
    Fragments Fragments Fragments

    Probe Radioactively-labeled None


    DNA or RNA
    Visualization Radioactivity from DNA Stain allows
    of DNA probe exposes X-ray shows all fragments
    Fragments film, subset of on gel
    fragments observed
    Non-isotopic Detection of DNA
    These directions REPLACE those
    found on 3-86  3-89
    • Place the membrane with the DNA side up in
    100 ml of Blue-Blot solution.
    • Soak the membrane at room temperature for
    10-15 minutes.
    • Remove the membrane with forceps and rinse
    in 200 ml distilled water.
    • Replenish the distilled water 3-4 times or until
    the membrane is destained and DNA bands
    are clearly visible.

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