Agarose Gel Electrophoresis and Southern Transfer

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Agarose Gel Electrophoresis and Southern Transfer
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Broad and Long Term Objective
To determine the copy number of Myb transcription
factor genes in the genome of the model plant
Arabidopsis thaliana
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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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Todays Laboratory Objectives

• To become familiar with a Southern
  Hybridization/Analysis
• - mechanics of setting up a Southern
• - 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 Analysis

• Definition
• Southern analysis is the transfer of denatured
  DNA from an agarose gel
• to a nylon or nitrocellulose membrane. This
  membrane is then probed
• with a complementary DNA or RNA fragment that
  has been radioactively
• or non-radioactively labeled.
• Plan
• 1. agarose gel electrophoresis
• 2. 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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Electrophoresis of genomic DNA
Odd numbered lanes contain undigested genomic
DNA Even numbered lanes contain digested genomic
DNA Your gel partial or complete digest of
genomic DNA?
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Separation of DNA fragmentsand preparation for
capillary transfer

• 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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DNA Transfer Accomplished via Capillary Action

• DNA transfer setup shown above
• DNA transfer is complete after 12-16 hours
• Electroblotting and vacuum blotting are
  alternative, more rapid DNA transfer techniques

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Fixation of DNA to the membrane

• After capillary transfer, single stranded DNA is
  loosely bound to the
• nylon/nitrocellulose membrane by hydrophobic
  interactions between
• nonpolar regions of the nylon and the exposed
  bases
• Hydrophobic interactions can be strengthened by
  removing water from the
• membrane (baking or microwaving the membrane)
• DNA can be covalently linked to nylon membranes
  by UV crosslinking (bases
• covalently bind to nylon amino groups)

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Next Week

• PCR will be employed to create a
• non-radioactively labeled Myb61 probe
• Probes will be hybridized to genomic DNA on
  the nylon membrane to determine which restriction
  fragment(s) may harbor the Myb genes