Southern Blotting DNA FingerprintingSouthern Blot A Southern Blot identifies specific sequences of DNA A Southern Blot may be used to determine a DNA fingerprint A Southern Blot may be used in forsenic medicineSouthern Blot Restriction Digest of DNA Electrophoresis Denaturation/Depurination Blotting Step ProbingdoneTo doTo doTo do 2 wksTodays laboratory: DNA fingerprinting in a hypothetical paternity determination. In this hypothetical case, DNA was extracted from samples obtained from two possible fathers, a mother and child. The DNA was were cleaved with a restriction enzyme.A Paternity CaseLoading Gels Each group should load their gel Run gel at approximately 125 volts for 45-60 minutes Lecture will be given while samples electrophorese.How to handle your gel after electrophoresisLoad Gels and then we will discuss recombinant DNA technologySample Volume Add 40 ul per well MUST WARM 5 min. at 65C Samples A. Standard DNA fragments B. Mother DNA cut with restriction enzyme C. Child DNA cut with restriction enzyme D. Possible Father #1 DNA with restriction enzyme E. Possible Father #2 DNA cut with restriction enzyme Depurination Step 8 minute (maximum) incubation in 100 ml .25N HCl Rinse gel several times with 100 ml distilled waterdepurinationDenaturation Step After rinsing gel with water Add 100 ml DNA denaturation solution 10 -15 min. Replace with fresh denaturation solution for 10-15 minutesGeneral Schematic for Southern Blot: DNASetting up the Southern Blot: pg. 3-82 Line a tray with plastic wrap Place “denatured” gel upside down on wrap Pre-wet nylon membrane in denaturation solution for 2-5 minutes Place nylon on top of inverted gelSetting up Southern Blot: pg. 3-82 Place filter paper on top of nylon membrane Remove air bubbles Place stack of paper towels on top of filter paper Place empty 400 ml beaker on towel Incubate overnightSetting up the Southern Blot transfer Plastic wrap Inverted Gel Nylon Membrane Filter paper Paper towels Weight Overnight incubation at room temperatureOvernight the ssDNA will diffuse by capillary transfer from gel onto nylon membrane!Next week we will stain this membrane for similarities between child and parents.Southern Blotting DNA FingerprintingAnalysis of Southern BlotA non-isotopic method of detectionRemember the Southern Blot requires that genomic DNA be first “digested” into smaller fragments that the DNA be separated on a gel that the DNA be denatured into single stranded DNASouthern Blot requires that you “probe” the fragments with a complementary sequence of DNA or RNA you have a means to “visualize” the binding between the probe and the DNAYou will probe the blot today Be sure and refer to your manual during these steps!You will probe the blot today First you must prepare the nytran membrane Use warm blocking buffer 45 minutes Remove blocking buffer Rinse container with water Add probe and mix well Incubate 10 minutesNext: many rinsing steps Rinse with 400 mls detection buffer for 10 min Rinse again with 200 mls for 15 min Rinse again with 200 mls for 15 min Be sure and agitate the solution with the buffer for complete rinsingColor Development I will prepare the substrate Add 8 mls of substrate Place DNA face down Place in 37C water bath Color should develop within 15-20 minhttp:/vector.cshl.org/resources/dnadetective.htmlLets look at some animations and examples of Southern Blots used in actual cases:http:/vector.cshl.org/resources/BiologyAnimationLibrary.htmAgarose gel electrophoresis Biotinylated DNA fragments labelled probes Avidin labelled enzyme Avidin has 4 binding sites for biotinBiotin-DNA binds to avidin labelled alkaline phosphatase.Substrate: 5-Bromo-4-chloro-3 indolyl phosphpate (BCIP)Product: Nitro Blue Tetrazolium to be reduced to an insoluble visible product.Southern Blot Analysis