Thermo Fisher Scientific Non-denaturing Agarose Gel Electrophoresis User guide

Brand
Thermo Fisher Scientific
Model
Non-denaturing Agarose Gel Electrophoresis
Type
User guide
Protocol
Non-denaturing Agarose Gel
Electrophoresis
Note
• Useaaskofatleastthreetimeslargervolumethan
thatofthesolutiontoavoidboilingover.
• Usethesame1Xelectrophoresisbuffertopreparethe
gelandtorunelectrophoresis.
• Dilute50XTAEBufferor10XTBEBuffertoa1X
concentrationimmediatelybeforeuse.
• UseTBEbufferforanalysisofDNAbandssmallerthan
1500bp.ForlargerDNA,useTAEbuffer.
• Forintensiedgelstaining,addethidiumbromideto
boththegelandtheelectrophoresisbufferatanal0.5
µg/mlconcentration.Alternatively,stainthegelafter
electrophoresis(seebelow).
Wear gloves when handling ethidium bromide.
• Forreliableanalysisofsupercoiled/relaxedplasmid
ethidiumbromideshouldnotbeincludedinthe
electrophoresisbufferorgel.Thegelshouldbestained
onlyafterelectrophoresisiscomplete.
• EthidiumbromideandexposuretoUVlightmaycause
DNAalterations.Therefore,avoidUVexposureanddo
notstainDNAwithethidiumbromideifthepuried
fragmentswillbeusedforcloningexperiments.
1. Weighouttherequiredamountofagarose
(dependingonthegelpercentage)intoan
Erlenmeyerask.
2. Addtheappropriatevolumeofeither1XTBEor
1XTAEbufferandswirltomix.
3. Weightheaskwiththesolution.
For high percentage gels (3-5%):addanexcessamountof
distilledwatertoincreasetheweightby10-20%.
4. Boilthemixtureinamicrowaveoven(atmedium
power)untiltheagarosemeltscompletely;swirl
theaskseveraltimeswhileboiling.Toprepare
thehighestqualityagarosegelsofanypercentage,
anadditional3-5minofboilingaftercompletely
meltingtheagaroseisrecommended.Asignicant
amountofwaterevaporatesduringthisprocedure
andthereforerestoringoftheinitialweight(instep
5)isrequiredtoobtainthedesiredpercentagegel.
5. Weightheaskagainandifnecessary,addhot
distilledwatertorestoretheinitialweight.
This protocol is for the Non-denaturing Agarose Gel Electrophoresis
North America
Technical Services:
techservice.genomics@thermofisher.com
Customer Services:
customerservice.genomics@
thermofisher.com
Tel 800 235 9880
Fax 800 292 6088
Europe and Asia
Technical Services:
techservice.emea.genomics@
thermofisher.com
Customer Services:
customerservice.emea.genomics@
thermofisher.com
thermoscientific.com
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Protocol
8. Immersethegelintothedesiredelectrophoresis
buffer.Loadthesamplesontothegel.
9. Runelectrophoresisat5-7V/cmuntilthe
bromophenolbluerunsapproximatelytwo-thirdsof
thewaydownthegel.
10. Afterelectrophoresisthegelcanbestainedby
immersingitintoa0.5µg/mlethidiumbromide
solutionfor15-20min,stainedwithSYBR®GreenI
oranyotherDNAstainingtechnique.
Warning. Hot agarose solution should be handled very
carefully.
For high percentage gels (3-5%):check(byweighing)that
theexcess10-20%ofwaterhasevaporatedand,if
needed,continueboilingtoremoveanyexcess,oraddhot
distilledwatertorestoretheinitialweight.
Optional: for intensied gel staining add ethidium bromide
to a nal concentration of 0.5 µg/ml. Mix well and heat for
1 min without boiling.
6. Coolthesolutionto65-70°C.Pourcarefullyona
cleancastingplatewithanappropriatecomb.If
bubblesappear,pushthemcarefullyawaytothe
sideswithapipettetip.
7. Solidifythegelforapproximately30minbefore
use.LowpercentageLMagarosegelsneedtobe
solidiedat4°C.
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Thermo Fisher Scientific Non-denaturing Agarose Gel Electrophoresis User guide

Brand
Thermo Fisher Scientific
Model
Non-denaturing Agarose Gel Electrophoresis
Type
User guide
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