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1、Southern Blotting DNA Fingerprinting,Southern Blot,A Southern Blot identifies specific sequences of DNAA Southern Blot may be used to determine a DNA fingerprintA Southern Blot may be used in forsenic medicine,Southern Blot,Restriction Digest of DNAElectrophoresisDenaturation/Depurination Blotting S
2、tepProbing,done,To do,To do,To do,2 wks,Todays 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 PaternityCase,L
3、oading Gels,Each group should load their gelRun gel at approximately 125 volts for 45-60 minutes Lecture will be given while samples electrophorese.,How to handle your gel after electrophoresis,Load Gels and then we will discuss recombinant DNA technology,Sample VolumeAdd 40 ul per wellMUST WARM 5 m
4、in.at 65C,SamplesA.Standard DNA fragmentsB.Mother DNA cut with restriction enzymeC.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 HClRinse gel severa
5、l times with 100 ml distilled water,depurination,Denaturation Step,After rinsing gel with waterAdd 100 ml DNA denaturation solution 10-15 min.Replace with fresh denaturation solution for 10-15 minutes,General Schematic for Southern Blot:DNA,Setting up the Southern Blot:pg.3-82,Line a tray with plast
6、ic wrapPlace“denatured”gel upside down on wrapPre-wet nylon membrane in denaturation solution for 2-5 minutesPlace nylon on top of inverted gel,Setting up Southern Blot:pg.3-82,Place filter paper on top of nylon membraneRemove air bubblesPlace stack of paper towels on top of filter paperPlace empty
7、400 ml beaker on towelIncubate overnight,Setting up the Southern Blot transfer,Plastic wrapInverted GelNylon MembraneFilter paperPaper towelsWeightOvernight incubation at room temperature,Overnight the ssDNA will diffuse by capillary transfer from gel onto nylon membrane!,Next week we will stain thi
8、s membrane for similarities between child and parents.,Southern Blotting DNA Fingerprinting,Analysis of Southern Blot,A non-isotopic method of detection,Remember the Southern Blot requires,that genomic DNA be first“digested”into smaller fragmentsthat the DNA be separated on a gelthat the DNA be dena
9、tured into single stranded DNA,Southern Blot requires that,you“probe”the fragments with a complementary sequence of DNA or RNAyou have a means to“visualize”the binding between the probe and the DNA,You will probe the blot today,Be sure and refer to your manual during these steps!,You will probe the
10、blot today,First you must prepare the nytran membraneUse warm blocking buffer 45 minutesRemove blocking bufferRinse container with waterAdd probe and mix wellIncubate 10 minutes,Next:many rinsing steps,Rinse with 400 mls detection buffer for 10 minRinse again with 200 mls for 15 minRinse again with
11、200 mls for 15 minBe sure and agitate the solution with the buffer for complete rinsing,Color Development,I will prepare the substrateAdd 8 mls of substratePlace DNA face downPlace in 37C water bathColor should develop within 15-20 min,http:/vector.cshl.org/resources/dnadetective.html,Lets look at s
12、ome animations and examples of Southern Blots used in actual cases:,http:/vector.cshl.org/resources/BiologyAnimationLibrary.htm,Agarose gel electrophoresis,Biotinylated DNA fragments labelled probesAvidin labelled enzymeAvidin has 4 binding sites for biotin,Biotin-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,
