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  3. Non-denaturing Agarose Gel Electrophoresis

Thermo Fisher Scientific Non-denaturing Agarose Gel Electrophoresis User guide

  • Hello! I've reviewed the user guide for Non-denaturing Agarose Gel Electrophoresis. This document provides detailed instructions for conducting non-denaturing agarose gel electrophoresis, including information on preparing the gel, selecting the right buffer, and staining techniques. It also offers important notes on handling ethidium bromide and preventing DNA alterations. I'm here to help with any questions you might have about using this protocol.
  • What buffer should I use for DNA bands smaller than 1500 bp?
    How should I stain the gel for intensified staining?
    What should I do if I need to analyze supercoiled/relaxed plasmids?
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
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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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