dna restriction analysis

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DNA Restriction Analysis Liam Gibbs Honors Biology May 20, 2016 Period 3 Introduction Gel Electrophoresis is the use of restriction enzymes to determine patterns in DNA. Restriction enzymes are a class of enzymes, catalyst that cut down DNA molecules when they recognize a repeating pattern. This is extremely important in the biotechnology world today. For example, the restriction enzyme BamH1 cuts DNA wherever recognizes the pattern of base pairs GGATCC. Since e
  DNA Restriction AnalysisLiam GibbsHonors BiologyMay 20, 2016Period   !ntrod ction Gel #lectro$%oresis is t%e se o& restriction en'ymes to determine $atterns in DNA( Restriction en'ymes are a class o& en'ymes, catalyst t%at c t do)n DNA molec les )%en t%ey recogni'e a re$eating $attern( *%is is e+tremely im$ortant in t%e biotec%nology )orld today( or e+am$le, t%e restriction en'yme BamH1 c ts DNA )%ere-er recogni'es t%e $attern o& base $airs GGA*..( /ince eac% being %as a di&&erent se ence o& base $airs, t%e lengt%s o& t%e &ragments )ill only matc% t%at $ersons o)n DNA( *%e en'yme )ors by acting lie a scanner, e+amining t%e DNA in searc% &or a $artic lar se ence( Ho)e-er, scientists )%o se restriction en'ymes %a-e a -ariety o& ses &or t%em( *%ey are also sed in criminal cases )%ere DNA is &o nd and can be sed to &ind a s s$ect( Restriction en'ymes can also be sed in $aternity cases to &ind t%e &at%er o& a c%ild( Gel #lectro$%oresis is %o) t%ese restriction en'ymes can be $ t to se( 3%en a sam$le o& DNA is collected, it is $reser-ed and d $licated in order to a-oid t%e c%ance o& t%e s$ecimen  being damaged( !t is t%en dyed and, e-al ated in a gel along )it% ot%er sam$les o& DNA( *%e gel is a strong s bstance, b t allo)s t%e &ragments to mo-e t%ro g% it( *%e gel is $laced in )ater( !n t%e &irst )ell t%ere is t%e ntainted sam$le o& t%e DNA( *%e ot%er )ells )o ld be &illed )it% t%e DNA o& s s$ects( #ac% sam$le )o ld %a-e been mi+ed )it% t%e same restriction en'yme( *%e )ater is t%en c%arged, $ositi-ely and negati-ely( *%e DNA &ragments )ill mo-e to)ards t%e $ositi-e c%arge( *%e smaller &ragments mo-ing & rt%er t%an t%e larger &ragments( !& t%ere is a matc% in t%e &ragments t%e s s$ect is most liely g ilty, 4 st as i& t%e &ragments )ere matc%ed in a $aternity case( *%e &irst $ r$ose o& t%is lab )as t%at )e )anted to see i& di&&erent restriction en'ymes c tDNA into di&&erent si'ed &ragments( Anot%er $ r$ose o& t%is lab )as to get $ractice )it% restriction en'ymes and gel electro$%oresis( inally t%e last $ r$ose )as to create a logarit%mic gra$% )it% no)n data to &ig re o t t%e ot%er lengt%s o& o r DNA &ragments( *%is ga-e s  $ractice )it% e+cel and creating tables( !n t%is e+$eriment t%e srcinal DNA is t%e control gro $, and t%e &inal si'e o& its &ragments is t%e inde$endent -ariable( *%e &inal si'es o& t%e DNA &ragments o& t%e ot%er sam$les are t%e de$endent -ariables( My %y$ot%esis &or t%is e+$eriment is t%at i& )e tae &o r di&&erent restriction en'ymes to c t t%e lambda DNA t%en )e )ill see di&&erent lengt%s o& t%e DNA &ragments(Materials5 Agarose Gel  5 *B# B &&er /ol tion 5 Lambda DNA 5 Restriction #n'ymes #coR!, BamH!, Hind!!!7 5 Plastic $i$ettes5 $i$ette ti$s 5 Hot Plate 5 #$$indor& reaction t bes 5 80 mL beaers 5 1000 mL &las 5 #lectro$%oresis c%amber 5 Grad ated .ylinder 5 Microcentri& ge 5 9orte+ 5 #t%idi m Bromide /tain 5 Loading Dye 5 /taining trays Proced reA: set $ restriction digest1(Label &o r 1(8 ml t bes in )%ic% yo )ill $er&orm restriction reactions: B &or BamH!, # &or #coR! &or Hind!!!, and &or no en'yme(2(;se table belo) as a c%eclist )%ile adding reagents to eac% reaction( Read do)n eac% col mn, adding t%e same reagent to all a$$ro$riate t bes< se a &res% ti$ &or eac% reagent( All gro $s s%are t%e same BamH!, #coR!, Hind!!! en'ymes at a central station((Pool and mi+ reagents by ta$$ing t%e t be bottom on lab benc%, or )it% a s%ort $ lse in microcentri& ge(=(!nc bate all reaction t bes &or a minim m o& 20 min tes at > degrees .elsi s( ?o r teac%er may instr ct yo to inc bate t%e reactions &or longer(  B: .ast Agarose Gel1( /eal ends o& gel@casting tray )it% ta$e, and insert )ell &orming comb( Place gel@casting tray o t o& t%e )ay on lab benc% so t%at agarose $o red in ne+t ste$ can set ndist rbed(2( .are& lly $o r eno g% agarose sol tion into casting tray to &ill to de$t% o& abo t 8mm( Gel s%o ld co-er only abo t 1 t%e %eig%t o& comb teet%( ;se a $i$et ti$ or toot%$ic to mo-e large b bbles or solid debris to sides or end o& tray )%ile gel is stillli id(( Gel )ill become clo dy as it solidi&ies( D not mo-e or 4ar casting tray )%ile agaroseis solidi&ying(=( 3%en agarose %as set, nseal ends o& casting tray( Place tray on $lat&orm or &ell bo+ so t%at comb is at negati-e(8( ill bo+ )it% tris@borate@#D*A *B#7 b &&er, to le-el t%at 4 st co-ers entire s r&ace o& gel(6( Gently remo-e comb, do not ri$ )ells>( Mae certain t%at sam$le )ells le&t by comb are com$letely s bmerged( !& dim$les are noticed aro nd t%e )ells slo)ly add b &&er ntil t%ey disa$$ear(C( *%e gel is no) ready to load )it% DNA( !& yo )ill be loading t%e gel d ring anot%er  $eriod, yo r teac%er )ill instr ct yo to co-er t%e electro$%oresis tan to $re-ent drying o& t%e gel(.: Load Gel1(Add 1 l loading dye to eac% reaction t be( Mi+ dye )it% digested DNA by ta$$ing t be on lab benc%, or )it% a $ lse in microcentri& ge(2(;se micro$i$ette to load contents o& eac% reaction t be into a se$arate )ell in gel, alignedas ill strated in ideal restriction digest o& lambda DNA( ;se a &res% ti$ &or eac% reaction t be(a(/teady $i$et o-er )ell sing t)o %ands( b(Be care& l to e+$el any air in micro$i$et ti$ end be&ore loading gel( !& air b bble &orms ca$ o-er )ell, DNA loading dye )ill &lo) into b &&er aro nd edges o& t%e )ell(7c(Di$ $i$et ti$ t%ro g% s r&ace o& b &&er, $osition it o-er t%e )ell, and slo)ly e+$el t%e mi+t re( / crose in t%e loading dye )eig%s do)n t%e sam$le, ca sing it to sin to t%e  bottom o& t%e )ell( Be care& l not to $ nc% ti$ o& $i$et t%ro g% bottom o& gel(D: #lectro$%orese 1(.lose to$ o& electro$%oresis c%amber and connect electrical leads to an a$$ro-ed  $o)er s $$ly, anode to anode red@red7 and cat%ode to cat%ode blac@blac7( Mae s re bot% electrodes are connected to same c%annel o& $o)er s $$ly( 2(* rn $o)er s $$ly on and set -oltage as directed by yo r instr ctor( /%ortly a&ter c rrent is a$$lied, loading dye can be seen mo-ing t%ro g% gel to)ard t%e $ositi-e  $ole o& electro$%oresis a$$arat s(
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