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Southern Transfer

Southern Transfer. 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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Southern Transfer

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  1. Southern Transfer

  2. 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

  3. Broad Overall Objective Is Myb61 a single or multicopy gene in A. thaliana

  4. Today’s 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

  5. 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)

  6. 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)

  7. 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

  8. Electrophoresis of Genomic DNA • Lambda Marker • Odd numbered lanes contain undigested genomic DNA • Even numbered lanes contain digested genomic DNA

  9. 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

  10. 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

  11. 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

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