Agarose. Gel. Electrophoresis.pdf.docx..

Electrophoresis

1. AgaroseGelElectrophoresis
Submittedby:AliRazaandAllahDitta
Submittedto:Dr.NasirMahmood
RollNo:538(046330),2405
Govt.IslamiaGraduateCollege,CivilLines,Lahore.

2. AgaroseGelElectrophoresis
Definition:
●Agarosegelelectrophoresisisamethodofgelelectrophoresis
usedinbiochemistry,molecularbiology,genetics,andclinical
chemistrytoseparateamixedpopulationofmacromoleculessuch
asDNA,RNAorproteinsinamatrixofagarose.
●Agaroseisanaturallinearpolymerextractedfromseaweedthat
formsagelmatrixbyhydrogen-bondingwhenheatedinabuffer
andallowedtocool.
●Theyarethemostpopularmediumfortheseparationofmoderate
andlarge-sizednucleicacidsandhaveawiderangeofseparation.
ChrisRocktoDirectMartinLutherKingJr.Biopic,SpielbergProducing|
Principle:
GelelectrophoresisseparatesDNAfragmentsbysizeinasolidsupport
mediumsuchasanagarosegel.Sample(DNA)arepipettedintothe
samplewells,followedbytheapplicationofanelectriccurrentwhich
causesthenegatively-chargedDNAtomigrate(electrophorese)towards
theanodal,positive(+ve)end.Therateofmigrationisproportionaltosize:
smallerfragmentsmovemorequicklyandwindupatthebottomofthegel.
DNAisvisualisedbyincludinginthegelanintercalatingdye,ethidium
bromide.DNAfragmentstakeupthedyeastheymigratethroughthegel.
Illuminationwithultravioletlightcausestheintercalateddyetofluoresce.
Thelargerfragmentsfluorescemoreintensely.Althougheachofthe
fragmentsofasingleclassofmoleculeispresentinequimolarproportions,
thesmallerfragmentsincludelessmassofDNA,takeuplessdye,and
thereforefluorescelessintensely.A“ladder”setofDNAfragmentsof
knownsizecanberunsimultaneouslyandusedtoestimatethesizesofthe
otherunknownfragments.

3. Requirements/InstrumentationofAgaroseGel
Electrophoresis
Theequipmentandsuppliesnecessaryforconductingagarosegel
electrophoresisarerelativelysimpleandinclude:
1.Anelectrophoresischamberandpowersupply
2.Gelcastingtrays,whichareavailableinavarietyofsizesand
composedofUVtransparentplastic.Theopenendsofthetrays
areclosedwithtapewhilethegelisbeingcast,thenremovedprior
toelectrophoresis.
3.Samplecombs,aroundwhichmoltenmediumispouredtoform
samplewellsinthegel.
4.Electrophoresisbuffer,usuallyTris-acetate-EDTA(TAE)or
Tris-borate-EDTA(TBE).
5.Loadingbuffer,whichcontainssomethingdense(e.g.glycerol)to
allowthesampleto“fall”intothesamplewells,andoneortwo
trackingdyes,whichmigrateinthegelandallowvisualmonitoring
ofhowfartheelectrophoresishasproceeded.
6.Staining:DNAmoleculesareeasilyvisualisedunderanultraviolet
lampwhenelectrophoresedinthepresenceoftheextrinsicfluor
ethidiumbromide.Alternatively,nucleicacidscanbestainedafter
electrophoreticseparationbysoakingthegelinasolutionof
ethidiumbromide.WhenintercalatedintodoublestrandedDNA,
fluorescenceofthismoleculeincreasesgreatly.Itisalsopossible
todetectDNAwiththeextrinsicfluor1-anilino8-naphthalene
sulfonate.
7.Transilluminator(anultravioletlightbox),whichisusedto
visualisestainedDNAingels.

4. StepsInvolvedinAgaroseGelElectrophoresis
1.Topreparegel,agarosepowderismixedwithanelectrophoresis
buffertothedesiredconcentration,andheatedinamicrowave
oventomeltit.
TheconcentrationofAgaroseGel
●Thepercentageofagaroseuseddependsonthesizeoffragments
toberesolved.
●Theconcentrationofagaroseisreferredtoasapercentageof
agarosetovolumeofbuffer(w/v),andagarosegelsarenormally
intherangeof0.2%to3%.
●Thelowertheconcentrationofagarose,thefastertheDNA
fragmentsmigrate.
●Ingeneral,iftheaimistoseparatelargeDNAfragments,alow
concentrationofagaroseshouldbeused,andiftheaimisto
separatesmallDNAfragments,ahighconcentrationofagaroseis
recommended.
2.Ethidiumbromideisaddedtothegel(finalconcentration0.5
ug/ml)tofacilitatevisualisationofDNAafterelectrophoresis.
3.Aftercoolingthesolutiontoabout60oC,itispouredintoacasting
traycontainingasamplecombandallowedtosolidifyatroom
temperature.
4.Afterthegelhassolidified,thecombisremoved,takingcarenotto
ripthebottomofthewells.
5.Thegel,stillinaplastictray,isinsertedhorizontallyintothe
electrophoresischamberandiscoveredwithabuffer.
6.SamplescontainingDNAmixedwithaloadingbufferarethen
pipettedintothesamplewells,thelidandpowerleadsareplaced
ontheapparatus,andacurrentisapplied.
7.Thecurrentflowcanbeconfirmedbyobservingbubblescoming
offtheelectrodes.
8.DNAwillmigratetowardsthepositiveelectrode,whichisusually
colouredred,inviewofitsnegativecharge.
9.ThedistanceDNAhasmigratedinthegelcanbejudgedby
visuallymonitoringmigrationofthetrackingdyeslikebromophenol
blueandxylenecyanoldyes.

5. ApplicationsofAgaroseGelElectrophoresis
Agarosegelelectrophoresisisaroutinelyusedmethodforseparating
proteins,DNAorRNA.
●EstimationofthesizeofDNAmolecules
●AnalysisofPCRproducts,e.g.inmoleculargeneticdiagnosisor
geneticfingerprinting
●SeparationofrestrictedgenomicDNApriortoSouthernanalysis,
orofRNApriortoNorthernanalysis.
●Theagarosegelelectrophoresisiswidelyemployedtoestimate
thesizeofDNAfragmentsafterdigestionwithrestrictionenzymes,
e.g.inrestrictionmappingofclonedDNA.
●Agarosegelelectrophoresisiscommonlyusedtoresolvecircular
DNAwithdifferentsupercoilingtopology,andtoresolvefragments
thatdifferduetoDNAsynthesis.
●Inadditiontoprovidinganexcellentmediumforfragmentsize
analyses,agarosegelsallowpurificationofDNAfragments.Since
purificationofDNAfragmentssizeseparatedinanagarosegelis
necessaryforanumberofmoleculartechniquessuchascloning,it
isvitaltobeabletopurifyfragmentsofinterestfromthegel.
AdvantagesofAgaroseGelElectrophoresis
●Formostapplications,onlyasingle-componentagaroseisneeded
andnopolymerizationcatalystsarerequired.Therefore,agarose
gelsaresimpleandrapidtoprepare.
●Thegeliseasilypoured,anddoesnotdenaturethesamples.
●Thesamplescanalsoberecovered.
DisadvantagesofAgaroseGelElectrophoresis
●Gelscanmeltduringelectrophoresis.
●Thebuffercanbecomeexhausted.
●Differentformsofgeneticmaterialmayruninunpredictableforms.