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Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Gel Electrophoresis of DNA,Molecular Genetics Presentation by:,Nana,Sugma,Mulyana,Febrina,Anggraini,Ginting,Faizal,Dony,Rifai,Nor,Aviva,Acknowledgements,Thanks go to Craig Millar,School of Biological Science,University of Auckland,Compiled by,Linda Macdonald,For NCEA Biology A.S.3.6,With support from the Royal Society Science,Mathematics&Technology Teacher Fellowship Scheme,What is Gel Electrophoresis,?,Electro=flow of electricity,phoresis,from the Greek=to carry across,A gel is a colloid,a suspension of tiny particles in a medium,occurring in a solid form,like gelatin,Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied,Charged particles can include DNA,amino acids,peptides,etc,Why do gel electrophoresis,?,When DNA is cut by restriction enzymes,the result is a mix of pieces of DNA of different lengths,It is useful to be able to separate the pieces-I.e.for recovering particular pieces of DNA,for forensic work or for sequencing,What is needed,?,Agarose,-a polysaccharide made from seaweed.Agarose is dissolved in buffer and heated,then cools to a gelatinous solid with a network of crosslinked molecules,Some gels are made with,acrylamide,if sharper bands are required,Buffer,-in this case TBE,The buffer provides ions in solution to ensure electrical conductivity.,Not only is the agarose dissolved in buffer,but the gel slab is submerged(submarine gel)in buffer after hardening,Also needed are a,power supply,and a,gel chamber,Gel chambers come in a variety of models,from commercial through home-made,and a variety of sizes,How does it work,?,DNA is an organic acid,and is,negatively,charged,(remember,D,N,A for,N,egative),When the DNA is exposed to an electrical field,the particles migrate toward the,positive,electrode,Smaller pieces of DNA can travel further in a given time than larger pieces,A gel being run,Agarose block,Positive electrode,DNA loaded in,wells in the agarose,Black background,To make loading wells easier,Comb,Buffer,Steps in running a gel,DNA is prepared by digestion with,restriction enzymes,Agarose is made to an appropriate thickness(the higher the%agarose,the slower the big fragments run)and,melted,in the microwave,The gel chamber is set up,the comb is inserted,The agarose may have a DNA dye added(or it may be stained later).The agarose is poured onto the gel block and cooled,The comb is removed,leaving little wells and buffer is poured over the gel to cover it completely,The DNA samples are mixed with a dense loading dye so they sink into their wells and can be seen,The DNA samples are put in the wells with a micropipette.,Micropipettes have disposable tips and can accurately measure 1/1,000,000 of a litre,Next,?,The power source is turned on and the gel is run.The time of the run depends upon the amount of current and%gel,and requires experimentation,At the end of the run the gel is removed(it is actually quite stiff),The gel is then visualized-UV light causes the bands of DNA to fluoresce,Animation,1,Simple,2,More,A gel as seen under UV light-some samples had 2 fragments,of DNA,while others had none or one,More,Many samples can be run on one gel-but it is important to keep track,Most gels have one lane as a DNA ladder-DNA fragments of known size are used for comparison,Still more,.,The DNA band of interest can be cut out of the gel and the DNA extracted-,Or DNA can be removed from the gel by,Southern Blotting,References,-presentations,Kreuzer,H.,Massey,A.,2001,Recombinant DNA and Biotechnology,2nd ed.,ASM Press,Washington,Turner,P.C.,et al,1997,Instant Notes in Molecular Biology,Bios,Oxford,Photos-L.D.Macdonald,2003,
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