Southern Transfer

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

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

Today’s Laboratory Objectives 1. To become familiar with a Southern Hybridization/Analysis a. mechanics of setting up a Southern a. mechanics of setting up a Southern b. What kinds of information can be gleaned b. What kinds of information can be gleaned 2. To be able to evaluate a restriction digest To distinguish between a partial and complete To distinguish between a partial and complete digest of genomic DNA using agarose gel digest of genomic DNA using agarose gel electrophoresis electrophoresis

Theoretical Basis of Southern Definition 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. 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 Plan 1. agarose electrophoresis 2. membrane transfer (capillary transfer) 3. detection (colorimetric) 1. agarose electrophoresis 2. membrane transfer (capillary transfer) 3. detection (colorimetric)

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)

Agarose Gel Electrophoresis 1.DNA fragments separated via agarose gel electrophoresis 2.Depurinate DNA - Remove adenine and guanine residues with HCl 3.Denature DNA - separate the DNA strands with NaOH 4. DNA neutralized w/ tris buffer

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

DNA Transfer Accomplished via Capillary Action  DNA transfer setup shown above  DNA transfer is complete after hours  DNA covalently linked to membrane via UV crosslinking or heating at 80 C for 2-4 hours

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

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