DNA Restriction Restriction Analysis Lab
Rylee Kopchak Biology Period 8 May 14, !1"
Introduction
#n this e$peri%ent, the scientists &sed restriction en'y%es to c&t DNA (ro% the bacteriophage la%bda into (rag%ents, )hich )ere then separated &sing gel electrophoresis* Restriction en'y%es, also kno)n as restriction endon&cleases, c&t a DNA %olec&le %olec&le at a partic&lar se+&ence kno)n as the recognition site* Restriction en'y%es are e$tre%ely i%portant tools (or analy'ing DNA* According to to Askabiologist.asu.edu, Askabiologist.asu.edu, -he en'y%e en 'y%e .scans/ a DNA %olec&le, looking (or a partic&lar se+&ence, &s&ally o( (o&r to si$ n&cleotides* 0nce it (inds this this recognition se+&ence, it stops and c&ts the strand* Restriction en'y%es ha2e a )idespread &se in %any %olec&lar genetics techni+&es s&ch as %apping DNA, 2eri(ying speci(ic DNA (rag%ents, and the generation o( reco%binant DNA %olec&les* Reco%binant DNA %olec&les are DNAs that consist o( genes (ro% t)o di((erent di((erent organis%s* Restriction en'y%es also play an i%portant role in identi(ying DNA strains o( a partic&lar species* Restriction 3rag%ent length poly%orphis% R3LP5 analysis is co%%only &sed to deter%ine a change in the genetic se+&ence o( DNA c&t by a restriction en'y%e* R3LP is &sed to identi(y indi2id&als indi2id&als in cri%inal and paternity cases, identi(y speci(ic %&tations, and trace inheritance patterns* patterns* 6el electrophoresis is a techni+&e &sed in the lab to separate charged %olec&les like DNA, RNA, and proteins according to to si'e* 6el electrophoresis )orks by sending an electric c&rrent across the gel so that one end has a positi2e charge, )hile the other has a negati2e charge* Molec&les %igrate to)ards the opposite charge7 (or e$a%ple, a %olec&le )ith a negati2e charge )o&ld be p&lled to)ard the positi2e positi2e end o( the gel* %aller %olec&les pass thro&gh thro&gh the gel at a +&icker rate, and there(ore, tra2el (&rther than larger %olec&les* 6el electrophoresis enables scientists to disting&ish DNA (rag%ents o( di((erent lengths* A(ter gel electrophoresis is cond&cted, (l&orescent or radioacti2e dyes are added, allo)ing scientists to st&dy the DNA* Agarose gels a co%ple$ s&gar %aterial5 are typically &sed a nd placed into an electrophoresis
tank, )here the electric c&rrent is sent thro&gh* -he p&rpose o( this lab lab )as to analy'e )hether di((erent restriction restriction en'y%es c&t DNA into into di((erent si'ed (rag%ents* #n addition, the scientists )anted to practice )orking )ith restriction en'y%es and gel electrophoresis* A(ter the e$peri%ent )as co%pleted, the scientists created a logarith%ic graph displaying the kno)n data5 in order to calc&late the lengths o( the re%aining DNA (rag%ents* Experiment Details
9ariable )as the restriction en'y%es en' y%es &sed to c&t c& t the Independent Variable Variable 7 -he #ndependent 9ariable DNA* Dependent Variable7 -he Dependent 9ariable ariable in this e$peri%ent )as the (rag%ent si'e and
distance the DNA (rag%ents tra2elled in the agarose gel* Control Group 7 -he control gro&p in this e$peri%ent )as the DNA that contained no restriction
en'y%e* Hypothesis 7 #t is hypothesi'ed that the restriction en'y%es )ill c&t the DNA into (rag%ents o(
2ario&s si'es, ca&sing the% to tra2el thro&gh the gel at di((erent rates* Materials • • • •
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Agarose gel -B: B&((er ol&tion La%bda DNA Restriction :n'y%es :coR1,
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Ba%;#, ;ind###5 Micropipettes Micropipette tips :ppendor( reaction t&bes
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:lectrophoresis cha%ber 6rad&ated cylinder Microcentri(&ge 9orte$ :thidi&% bro%ide stain Loading dye 6lo2es 6oggles taining trays =ltra2iolet light so&rce harpie
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Procedure
1* Label (o&r (o&r 1*<%L t&bes, in in )hich yo& yo& )ill per(or% per(or% restri restriction ction reactio reactions7 ns7 B (or BamHI (or BamHI , : (or EcoRI (or EcoRI , ; (or HindIII (or HindIII , and > (or no en'y%e* * =se table table belo) belo) as a checklist checklist )hile )hile adding reagents reagents to each each reaction reaction** Read do)n do)n each col&%n, adding the sa%e reagent to all appropriate t&bes? &se a (resh tip (or each reagent* Tu
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* Pool and %i$ %i$ reagents reagents by tapping tapping the t&be t&be botto% botto% on lab bench, bench, or )ith )ith a short p&lse p&lse in a %icrocentri(&ge* 4* #nc&bate #nc&bate all reactio reaction n t&bes (or (or a %ini%&% %ini%&% o( o( ! %in&tes %in&tes at at CE* <* Retrie Retrie2e 2e a pre%a pre%ade de agar agarose ose gel* gel* "* Add 1 @L loading loading dye dye to each reaction reaction t&be* t&be* Mi$ dye dye )ith digested digested DNA DNA by tapping tapping t&be on lab bench, or )ith a p&lse in the %icrocentri(&ge* C* =se %icropipe %icropipette tte to load load contents contents o( each each reaction reaction t&be into into a separate separate )ell in gel* gel* =se a (resh tip (or each reaction t&be* a* teady teady pipe pipett o2er o2er )ell )ell &sin &sing g t)o hands* hands* b* Be care(&l to e$pel any air in %icropipette tip end be(ore loading gel* c* Dip pipet tip thro&gh thro&gh s&r(ace s&r(ace o( b&((er b&((er,, position position it o2er o2er the )ell, )ell, and slo)ly slo)ly e$pel e$pel the %i$t&re* &crose in the loading dye )eighs do)n the sa%ple, ca&sing it to sink to the botto% o( the )ell* 8* Elose top top o( electrophor electrophoresis esis cha%ber cha%ber and and connect electri electrical cal leads to to an appro2ed appro2ed po)er s&pply, anode to anode redred5 and cathode to cathode blackblack5* blackblack5* Make s&re both electrodes are connected to sa%e channel o( po)er s&pply*
F* -&rn -&rn po)er s&pply on and set set 2oltage 2oltage as directed directed by yo&r instr& instr&ctor ctor** hortly hortly a(ter a(ter the c&rrent is applies, loading dye can be seen %o2ing thro&gh gel to)ard positi2e pole o( electrophoresis apparat&s* 1!* -he loading dye )ill )ill e2ent&ally resol2e into t)o bands o( color* -he (aster%o2ing, (aster%o2ing, p&rplish band is the dye bro%ophenol bl&e? the slo)er%o2ing, a+&a band in $ylene cyanol* Bro%ophenol bl&e %igrates thro&gh the the gel at the sa%e rate as a DNA (rag%ent appro$i%ately !! base pairs long* Gylene cyanol %igrates at a rate e+&i2alent to appro$i%ately !!! base pairs* 11* Allo) the DNA to electrophorese &ntil the bro%ophenol bro%ophenol bl&e band nears the end o( the gel* 1* -&rn o(( po)er s&pply, s&pply, disconnect disconnect leads (ro% the inp&ts, and re%o2e top o( electrophoresis cha%ber* 1* Eare(&lly re%o2e casting tray tray and slide gel into staining staining tray labeled )ith yo&r gro&p gro&p na%e* -ake -ake gel to yo&r instr&ctor (or staining* "esults •
H-his pict&re sho)s an ideal agarose gel a(ter gel electrophoresis* A(ter staining the dye )ith Bro%ophenol bl&e, the DNA (rag%ents beco%e 2isible &nder =9 light, and the scientist are able to see the distance each ea ch (rag%ent tra2elled*
Distanc Dist ance e Travelled Travelled by by ra!" ra!"ents ents #$t #$ t % it& 'ind 'ind 1.6 1.4 f(x) = - 0.01x + 1.9
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Distance Travelled by ra!"ent ("")
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H-his graph sho)s the distance tra2elled by the DNA (rag%ents c&t )ith ;ind### in %illi%eters* #n addition, it displays displays a line o( best (it and the correlation bet)een (rag%ent si'e and the distance it tra2eled thro&gh the gel* HindIII
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, ! C H-his chart displays the %eas&red distances tra2elled by DNA c&t )ith each restriction en'y%e7 ;ind###, :coR#, and Ba%;#* Ba%;#* #t also sho)s the the act&al n&%ber o( base pairs that (or%ed each (rag%ent, along )ith the n&%ber the scientist calc&lated in the e$peri%ent* Discussion •
A(ter co%pleting the e$peri%ent, the the scientist/s hypothesis )as s&pported* -he
restriction en'y%es did c&t the DNA into (rag%ents o( di((erent lengths, ca&sing the% to tra2el 2ario&s distances* -he restriction en'y%e ;ind### c&t the DNA into the %ost pieces se2en (rag%ents5, ca&sing the% to tra2el 1%% thro&gh the agarose gel* gel* :coR# c&t the DNA strand into si$ (rag%ents* As a res<, res<, the DNA tra2elled F*4%% thro&gh the gel* 3inally, Ba%;# c&t the DNA into into only (i2e (rag%ents, tra2elling C<%% thro&gh the gel* Restriction en'y%es c&t DNA at di((erent di((erent locations beca&se each o( the% has a distinct se+&ence o( n&cleotides that tells it )here to c&t the strand* -he %ore ti%es the restriction restriction en'y%e c&ts the DNA, the (arther it
)ill tra2el thro&gh the the gel d&ring gel electrophoresis* -his is beca&se s%aller s%aller (rag%ents %o2e thro&gh the gel easier and +&icker than larger DNA (rag%ents*
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o%e %istakes that %ay ha2e been %ade d&ring the e$peri%ent incl&de
%eas&re%ent errors, and %istakes %istakes %ade )hile staining the the gel* Mistakes in %eas&re%ent %ay %ay ha2e occ&rred )hile %eas&ring the a%o&nt o( restriction en'y%es and DNA to p&t in the reaction t&bes* #n addition, slightly inacc&rate %eas&re%ents co&ld ha2e been %ade )hen the scientist %eas&red the distance each DNA (rag%ent (rag%ent tra2elled thro&gh the gel* Beca&se o( this error, the calc&lated base pairs 2aried %arginally (ro% the act&al n&%ber o( base pairs* 3inally, a(ter dying the gel )ith Bro%ophenol bl&e, the the DNA did not appear &nder the light* -his %ay ha2e been beca&se the dye )as ine((ecti2e or the light so&rce )as not strong eno&gh* "esources •
DNA Restriction Analysis Analysis Lab Man&al
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https7IIaskabiologist*as&*ed&Irestrictionen'y%es
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http7II)))*yo&rgeno%e*orgI(acts http7II)))*yo&rgeno%e*orgI(actsI)hatisgelelectrophoresis I)hatisgelelectrophoresis
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