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Agarose Gel Electrophoresis and Southern Transfer

Agarose Gel Electrophoresis and Southern Transfer. Broad and Long Term Objective. To determine the copy number of Myb transcription factor genes in the genome of the model plant Arabidopsis thaliana. Research Plan. Isolate Genomic DNA. Digest Genomic DNA with Various Restriction Enzymes.

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Agarose Gel Electrophoresis and Southern Transfer

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  1. Agarose Gel Electrophoresis and Southern Transfer

  2. Broad and Long Term Objective To determine the copy number of Myb transcription factor genes in the genome of the model plant Arabidopsis thaliana

  3. 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

  4. Today’s 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

  5. 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)

  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. 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?*

  8. 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

  9. 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

  10. 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)

  11. 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

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