Southern Transfer

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Southern Transfer
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Research Plan
Isolate Genomic DNA
Digest Genomic DNA with Various Restriction
Enzymes
Agarose Gel Electrophoresis and Southern Transfer
Southern Blot
Make Non-Radioactive Myb Probe
Hyribidize Probe to Southern Blot
Washes and Colorimetric Detection
Data Analysis
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Broad Overall Objective

• Is Myb61 a single or multicopy gene in A.
  thaliana

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Todays Laboratory Objectives

• To become familiar with a Southern
  Hybridization/Analysis
• a. mechanics of setting up a Southern
• b. What kinds of information can be
  gleaned
• To be able to evaluate a restriction digest
• To distinguish between a partial and
  complete
• digest of genomic DNA using
  agarose gel
• electrophoresis

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Theoretical Basis of Southern

• Definition
• Southern analysis is the transfer of
  denatured DNA form an agarose gel to a membrane
  on which it can be analysed using a labelled
  complementary DNA or RNA probe.
• Plan
• 1. agarose electrophoresis2. membrane transfer
  (capillary transfer)3. detection (colorimetric)

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Loading the Agarose Gel

• Lane 1 Lambda Hind III Marker (negative control
• Lane 2 Genomic DNA/Bam HI
• Lane 3 Genomic DNA/Eco RI
• Lane 4 Genomic DNA/Hind III
• Lane 5 Genomic DNA/PST I
• Lane 6 Genomic DNA/Eco RI Pst I
• Lane 7 Genomic DNA/Bam HI Hind III
• Lane 8 Myb61 cDNA clone (positive control)

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Agarose Gel Electrophoresis

• DNA fragments separated via agarose
• gel electrophoresis
• Depurinate DNA - Remove adenine and guanine
  residues with HCl
• Denature DNA - separate the DNA strands with
  NaOH
• 4. DNA neutralized w/ tris buffer

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Electrophoresis of Genomic DNA

• Lambda Marker
• Odd numbered lanes contain undigested genomic DNA
• Even numbered lanes contain digested genomic DNA

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DNA Transfer Accomplished via Capillary Action

• DNA transfer setup shown above
• DNA transfer is complete after 12-16 hours
• DNA covalently linked to membrane via UV
  crosslinking or heating at 80 C for 2-4 hours

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Hybridization and Detection
Prehybridized at 60 C in seal-a-meal bag to mask
free potential DNA binding site on membrane that
could non-specifically bind probe
molecules Membrane hybridized to labelled myb61
probe molecules at 60 C Membrane is washed and
chemiluminescent detection is performed to
identify fragments that are complementary in
sequence to the metacaspase probe
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Next Week

• PCR will be employed to create
  non-radioactively labelled myb61 probe
• Homologous probe will be generated
• Probes will be hybridized to genomic DNA on
  membrane to determine which restriction
  fragment(s) may harbor the Myb61 gene