Gel Electrophoresis Lab Isabella Haberstock Honors Biology May 20, 2016 Pd !
Introduction
In this lab, "e #sed three di$$erent restriction en%y&es on la&bda '() to see ho" each "o#ld c#t #p the '() into $rag&ents *estriction en%y&es are en%y&es that each only "ork $or a certai certain n '() se+#en se+#ence, ce, "hich is called called a recogni recognitio tion n site site (5) nce the en%y&e $inds its se+#e se+#enc ncee in the the '(), '(), it c#ts c#ts the the '() '() in bet" bet"ee een n t"o t"o n#cle n#cleoti otide dess to crea create te $rag& $rag&ent ents s 'epending on "here the certain se+#ences are, the '() $rag&ents can be &any di$$erent si%es bac teria, "hich #ses the en%y&es to kill -ir#ses (5) (4) *estriction en%y&es are $o#nd in bacteria, *estriction en%y&es are one o$ the $o#ndations $or biotechnology research .cientists can #se the& to clone '(), create a '() $ingerprint, present as $orensic e-idence, and help in paternity cases (5) .cientists can also #se restriction en%y&es to c#t a '() se+#ence in a certain place so they can insert a $oreign '() $rag&ent into the se+#ence )n e/a&ple o$ this is that scientists can introd#ce a ne" '() se+#ence into an Ecoli bacterias geno&e and prod#ce ins#lin proteins, "hich are then #sed to help people "ith diabetes ) geno&e is the part o$ an organis&s organis&s '() that holds instr#ctions that &ake e- ery part o$ a gro"ing being $#nction aking aking ad-antage o$ restriction en%y&es and certain genes is called gene technology, and &aking ins#lin is one o$ &any e/a&ples (3) )s $or paternity and cri&inal cases, a ðod o$ identi$ication called *3LP 4restriction $rag&ent length poly&orphis&5 is #sed *3LP is the di$$erence in '() $rag&ents "hen the '() has been c#t by restriction en%y&es o&paring the cri&e scene sa&ple '() $ingerprint and those o$ any possible s#spects lead a scientist to "ho co&&itted a cri&e his sa&e ðod is also #sed to $ind the $ather o$ a baby (6) ne "ay to #se *3LP is "ith "ith gel electr electroph ophore oresis sis Gel electrop electrophor horesi esiss is #sing #sing an electrical c#rrent to sort '() $rag&ents by si%e ne end o$ a gel has &any "ells in it .cientists
insert '() co&bined "ith a restriction en%y&e into these "ells '() is negati-ely charged, so a negati-e charge sho#ld be applied on the side o$ the gel containing '() he other side o$ the gel is positi-ely charged he '() "ill nat#rally be attracted to the positi-e and "ill &o-e thro#gh the gel he restriction en%y&es c#t the '() into di$$erent si%e $rag&ents, and the shorter pieces "ill &o-e thro#gh the gel $aster than the longer pieces he banded pattern created by the pieces are a '() $ingerprint $ingerprint hese can be #sed $or -ario#s types o$ identi$ication (1) he p#rpose o$ this lab is to $ind o#t i$ di$$erent restriction en%y&es "ill c#t the sa&e type o$ '() into the sa&e si%e $rag&ents or di$$erent si%e $rag&ents his lab is also to get practice doing gel electrophoresis and to beco&e $a&iliar "ith restriction en%y&es en% y&es he lab also re+#ired a logarith&ic graph that "as #sed to $ind in$or&ation on the '() $ingerprints, "hich is practice $or #sing gi-en in$or&ation to obtain the rest o$ the in$or&ation in$or&ation he dependent -ariable in this lab is the '() $ingerprints created by the banded patterns he independent -ariable is the restriction en%y&es he control gro#p in this e/peri&ent is the test t#be containing the la&bda '() "ith "ater I$ "e #se three di$$erent restriction en%y&es to c#t la&bda '(), then each en%y&e "ill c#t the '() into di$$erent si%e $rag&ents Materials • • • • • • • • • • •
3o#r 17 &L t#bes *estriction en%y&es8 Bam en%y&es8 BamHI, HI, Eco Eco*I, *I, Hind Hind III III 9ater 9a ter $or control t#be Hot "ater bath Micropipette La&bda '() risborateE') b#$$er Microcentri$#ge Loading dye Gel casting tray and "ell$or&ing co&b ape
• • •
)garose Electrophoresis bo/ Po"er s#pply
Procedures
1 Label $o#r 17&L t#bes, t#bes, in "hich "hich yo# "ill per$or& restriction restriction reactions8 B $or Bam $or BamHI, HI, E $or Eco $or Eco*I, *I, H $or Hind $or Hind III, III, and : $or no en%y&e 2 ;se the table table belo" as a checklist checklist "hile adding adding reagents to each reaction reaction *ead do"n each col#&n, adding the sa&e reagent to all the appropriate t#bes< #se a $resh tip $or each reagent )ll gro#ps share the sa&e Bam sa&e BamHI, HI, Eco Eco*I, *I, Hind Hind III III en%y&es at a central station Tube B E H :
DNA =>L =>L =>L =>L
Buffer 7>L 7>L 7>L 7>L
BamHI
EcoRI
1>L : : :
: 1>L : :
Hind III
: : 1>L :
H! : : : 1>L
! Pool and &i/ reagents by tapping the t#be botto& on lab bench, or "ith a short p#lse in a &icrocentri$#ge = Inc#bate all reaction t#bes $or a &ini&#& o$ 20 &ins at !?@ 7 .eal ends o$ gel casting casting tray "ith tape, and insert insert "ell$or&i "ell$or&ing ng co&b Place gel casting casting tray o#t o$ the "ay on lab bench, so that agarose po#red in the ne/t step can set #ndist#rbed 6 are$#lly are$#lly po#r eno#gh eno#gh agarose agarose sol#tion sol#tion into casting casting tray tray to $ill the the depth o$ abo#t abo#t 7&& Gel sho#ld co-er only abo#t 1A! the height o$ co&b teeth ;se a pipet tip or toothpick to &o-e large b#bbles or solid debris to sides or end o$ tray, "hen gel is still li+#id ? Gel "ill "ill beco&e clo#dy clo#dy as it solidi solidi$ies $ies 4abo#t 4abo#t 10 &ins5 &ins5 'o not &o-e &o-e or ar casting casting tray tray "hile agarose is solidi$ying C 9hen agarose agarose has set, set, #nseal ends ends o$ casting casting tray tray Place tray tray on plat$or& plat$or& o$ gel gel bo/, so that co&b is at negati-e 4black5 end D 3ill bo/ "ith "ith trisborate trisborateE' E') ) 4BE5 b#$$er b#$$er,, to a le-el that #st co-ers co-ers entire entire s#r$ace s#r$ace o$ gel 10 Gently re&o-e re&o-e co&b, taking taking care not to rip "ells
11 Make certain that sa&ple "ells le$t by co&b are co&pletely s#b&erged s#b&erged I$ di&plesF are noticed aro#nd "ells, slo"ly add b#$$er #ntil they disappear 12 he gel is no" ready ready to load "ith "ith '() 1! )dd 1>L loading loading dye to each reaction reaction t#be Mi/ dye "ith digested '() by tapping tapping t#be on lab bench, or "ith a p#lse in &icrocentri$#ge 1= ;se &icropipette to load contents o$ each reaction t#be into a separate separate "ell in gel, aligned as ill#strated in Ideal *estriction 'igest o$ la&bda '() 4 "35 ;se a $resh tip $or each reaction t#be a .teady .teady pipe pipett o-er o-er "ell "ell #sin #sing g t"o hands hands b Be care$#l to e/pel any air in &icropipette tip end be$ore loading gel 4I$ air b#bble $or&s capF o-er "ell, '()Aloading dye "ill $lo" into b#$$er aro#nd edges o$ "ells5 c 'ip pipet tip tip thro#gh thro#gh s#r$ace s#r$ace o$ b#$$er, b#$$er, positi position on it o-er the the "ell, and slo"ly slo"ly e/pel e/pel the &i/t#re .#crose in the loading dye "eighs do"n the sa&ple, ca#sing it to sink to the botto& o$ the "ell Be care$#l not to p#nch tip o$ pipet thro#gh botto& o$ gel 17 lose top o$ electrophoresi electrophoresiss cha&ber and connect electrical electrical leads to an appro-ed appro-ed po"er s#pply, anode to anode 4red to red5 and cathode to cathode 4black to black5 Make s#re both electrodes are connected to sa&e channel o$ po"er s#pply 16 #rn po"er s#pply on and set -oltage as directed by yo#r instr#ctor instr#ctor .hortly a$ter c#rrent c#rrent is appli applied ed,, load loadin ing g dye can can be seen seen &o-i &o-ing ng to"a to"ard rd gel to"a to"ard rd posi positi ti-e -e pole pole o$ electrophoresis apparat#s 1? he loadin loading g dye "ill "ill e-ent# e-ent#all ally y resolresol-ee into into t"o bands bands o$ color color he $aster $aster&o&o-ing ing,, p#rplish band is the dye bro&ophenol bl#e< the slo"er&o-ing, a+#a band is /ylene cyano cyanol l Bro& Bro&op ophe henol nol bl#e bl#e &igr &igrat ates es thro thro#gh #gh gel gel at sa&e sa&e rate rate as a '() '() $rag $rag&e &ent nt appro/i appro/i&at &ately ely !00 base pairs pairs long long ylene ylene cyanol cyanol &igrat &igrates es at a rate rate e+#i-al e+#i-alent ent to appro/i&ately 2000 base pairs
1C )llo" the '() to to electrophorese electrophorese #ntil the bro&ophenol bro&ophenol bl#e band nears the end o$ the gel o#r instr#ctor &ay &onitor the progress o$ electrophoresis in yo#r absence< in that case, o&it steps 1D and 20 1D 1D #rn #rn o$$ o$$ po"e po"err s#pp s#pply ly,, disc discon onne nect ct lead leadss $ro& $ro& the the inp# inp#t, t, and and re&o re&o-e -e top top o$ electrophoresis cha&ber 20 are$#lly are$#lly re&o-e casting casting tray and slide gel into staining staining tray labeled "ith yo#r gro#p na&e ake gel to yo#r instr#ctor $or staining () Results
he lab &an#al contained a pict#re o$ "hat o#r gel sho#ld ha-e looked like ("1) )ll distance &eas#re&ents "ere taken $ro& the ideal gel pict#re It contains all the correct '() $ingerprints $or Hind $or Hind III, Eco III, Eco*I, *I, Bam BamHI, HI, and the control gro#p #r gel did not rese&ble the ideal gel pict#re and did not ha-e any '() $ingerprints present Ideal $el and %ab $el
"1& his is a pict#re o$ "hat the ideal gel "o#ld look like 4le$t5 and o#r gel 4right5 he ideal gel sho"s ho" $ar the '() $rag&ents &o-ed $or each restriction en%y&e and the control gro#p his i&age sho"s the tr#e '() $ingerprint $or each en%y&e he act#al gel that res#lted $ro& this lab sho"s no '() $ingerprints
;sing the ideal gel pict#re abo-e ("1), "e took &eas#re&ents in &illi&eters $ro& the top o$ the gel to the top o$ each band $or the Hind the Hind III III en%y&e he kilo base pairs $or Hind $or Hind III III "ere incl#ded incl#ded in the lab &an#al, so there "as eno#gh in$or&ation in$or&ation to co&plete the graph belo" (") 9e #sed the in$or&ation that "e had abo#t Hind abo#t Hind III III to get this graph )$ter plotting the points, "e inserted a best $it line to get a linear e+#ation that "o#ld help #s $ig#re o#t the in$or&ation $or the other t"o restriction en%y&es
log of kilo base pairs by distance in mm 1.6 1.4 1.2
f(x) = - 0.01x 0.01x + 1.82
1
log of ase !ai"s
0.8 0.6 0.4 0.2 0 30
40
50
60
70
80
90
100
110
1 20
130
distance in mm "& his graph is a scatter plot containing the distance 4&&5 $ro& the top o$ the gel to each band and the log o$ the kilo base pairs he indi-id#al points on the scatter plot are $or the Hind III III restriction en%y&e, and a best $it line "as added to the graph to esti&ate the kilo base pairs o$ the other t"o restriction en%y&es
)$ter obtaining the linear e+#ation $ro& the graph abo-e ("), "e &eas#red the distances $or the other t"o restriction en%y&es #sing the ideal gel pict#re ("1) 9e pl#gged the distance
-al#es in $or x $or x in in the e+#ation to obtain the calc#lated kilo base pairs recorded in the table belo" ("3) he act#al n#&ber o$ kilo base pairs p airs "ere pro-ided by the teacher Distance and 'ilo Base Pairs for ac Restriction n*+,e III Hind III
'istance 4&&5
kilo base pairs
EcoRI
BamHI
'istance 4&&5
alc#lated kilo base pairs
)ct#al kilo base pairs
'istance 4&&5
alc#lated kilo base pairs
)ct#al kilo base pairs
!?
2?7
=0
1D1
2=C
=7
16!
16C
=2
2!1
==
16D
212
70
1=0
12!
76
D =
60
10!
? =
67
C C
? 2
67
6 6
?0
? 6
7 C
6?7
C 2
6 C
C0
= =
?2
? 1
7 6
?0
? 6
7 6
111
2 !
?7
6 7
= D
120
2 0
D0
= 1
! 7
"3& his table sho"s the distance 4&&5 $ro& the top o$ the gel to each part o$ the banded pattern, the esti&ated n#&ber o$ kilo base pairs, and the act#al n#&ber o$ kilo base pairs he distance "as &eas#red $ro& the ideal gel pict#re abo-e 4 "15 he calc#lated kilo base pairs "ere deter&ined by the linear e+#ation $or the best $it line in the graph abo-e 4 "5 he act#al kilo base pairs "ere pro-ided by the teacher a$ter the lab "as co&pleted
Discussion
he res#lts o$ this lab pro-ed that &y hypothesis "as correct8 each restriction en%y&e did create a di$$erent '() $ingerprint $or la&bda '() *estriction en%y&es do c#t '() into a -ariety o$ si%es beca#se the certain se+#ence that the en%y&e is looking $or co#ld appear any n#&ber o$ ti&es, and it does not ha-e to be per$ectly spaced o#t he restriction en%y&es "ill c#t '() in di$$erent di$$erent places regardless o$ ho" &any ti&es the re+#ired se+#ence appears he shorter '() $rag&ents did &o-e $arther thro#gh the gel than bigger pieces he a&o#nt that the $rag&ents &o-e is based on length he s&aller '() $rag&ents are easier to &o-e thro#gh the gel than longer pieces he spaces bet"een the bands in the '() $ingerpr $ingerprint int pro-e that the '() "as c#t into -arying si%es beca#se so&e &o-e &ore than others
here "ere &any so#rces o$ error that "ere present "hen "e did this lab he linear e+#ation e+#ation $ro& the logarith&ic logarith&ic graph (") "as not co&pletely acc#rate beca#se the best $it line did not to#ch e-ery single point on the scatter plot #r teacher ga-e #s the act#al base pair -al#es so "e co#ld see the di$$erence )nother error "as that "e did not ha-e access to a &icropipette and "e had to #se less acc#rate pipettes hey "ere not -ery e$$ecti-e and ca#sed so&e o$ the la&bda '() and restriction en%y&e &i/t#res to &iss the "ells in the gel and get &i/ed into the b#$$er b #$$er )nother big error "as that the gels "ere s#pposed to be r#n thro#gh the electrical c#rrent $or abo#t an ho#r and a hal$ 9e ran o#t o$ ti&e and only ran the gels $or t"enty &in#tes 9e also stained the& $or too long a$ter "e took the& o#t o$ the electrophoresis bo/ 9e had no ; light, "hich "as needed to properly see the banded pattern $or the type o$ dye "e #sed )ll o$ these $actors contrib#ted to "hy no '() $ingerprint "as present on o#r gel References
Biggs, )lton, )lton, 9hitn 9hitney ey rispi rispin n Hagins, Hagins, 9illi 9illia& a& G Hollid Holliday ay,, hris hris L Japick Japicka, a, Linda Linda 1- Biggs, L#ndgren, )nn Haley MacJen MacJen%ie %ie,, 9illi 9illia& a& ' *ogers, *ogers, Marion Marion B .e"er, .e"er, 'inah 'inah Kike Kike Glencoe Science Biology Biology ol#&b#s8 McGra"Hill o&panies, Inc, 2012 Print - 3reyer, Greg ) and 'a-id ) Mickles '() *estriction )nalysis )nalysis JitF B#rlington8 arolina
Biological .#pply o&pany, 1DD7 Print 3- Hendrickson, Jirstin he ;ses o$ *estriction En%y&esF Livestrong.com En%y&esF Livestrong.com 'e&and Media,
Inc, 1 #ly 2017 9eb 1C May 2016 En%y&esF ASU School of Life Sciences Sciences )ri%ona Board o$ *egents, 201= 9e 9eb b 4- *estriction En%y&esF ASU 1C May 2016
En%y&esF Biotech Learning Hub Hub he ;ni-ersity o$ 9aikato, 20 (o-e&ber 5- *estriction En%y&esF Biotech 200? 9eb 9eb 1C May Ma y 2016 6- *estriction 3rag&ent Length Poly&orphis&F NB! Poly&orphis&F NB! (BI, 201= 9e 9eb b 1D May Ma y 2016
