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 Agarose Gel Electrophoresis and Southern Transfer Make Non-Radioactive Myb Probe Hyribidize Probe to Southern Blot Washes and Colorimetric Detection Data Analysis Southern Blot

Today’s Laboratory Objectives 1. To become familiar with a Southern Hybridization/Analysis - mechanics of setting up a Southern - 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 Analysis Definition Definition Southern analysis is the transfer of denatured DNA from an agarose gel Southern analysis is the transfer of denatured DNA from an agarose gel to a nylon or nitrocellulose membrane. This membrane is then probed to a nylon or nitrocellulose membrane. This membrane is then probed with a complementary DNA or RNA fragment that has been radioactively with a complementary DNA or RNA fragment that has been radioactively or non-radioactively labeled. or non-radioactively labeled. Plan Plan 1. agarose gel electrophoresis 2. membrane transfer (capillary transfer) transfer) 3. detection (colorimetric) 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)

Odd numbered lanes contain undigested genomic DNA Even numbered lanes contain digested genomic DNA Your gel: partial or complete digest of genomic DNA? * Electrophoresis of genomic DNA

Separation of DNA fragments and preparation for capillary transfer 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

DNA Transfer Accomplished via Capillary Action  DNA transfer setup shown above  DNA transfer is complete after hours  Electroblotting and vacuum blotting are alternative, more rapid DNA transfer techniques

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)

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