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  3. Alkaline Agarose Gel Electrophoresis

Thermo Fisher Scientific Alkaline Agarose Gel Electrophoresis User guide

  • Hello! I've reviewed the user guide for Agarose Gel Electrophoresis and am ready to assist you with any questions you have about the procedure. This document contains instructions for preparing the gel, sample loading, running the electrophoresis, and staining procedures. Let me know how I can help!
  • Are double stranded DNA ladders recommended for denaturing electrophoresis?
    What is the recommended flask size for the solution?
    What should I do if bubbles appear when pouring the gel?
    What is the electrophoresis running voltage?
Protocol
Note
• DoublestrandedDNAladdersarenotrecommended
fordenaturingelectrophoresisastheymayforman
atypicalpattern.However,thesediscrepanciesare
normallyacceptableforanalysisofcDNAorother
ssDNAinalkalinegels.
• Useaaskofatleastthreetimeslargervolumethan
thatofthesolutiontoavoidboilingover.
• Weargloveswhenhandlingethidiumbromide.
1. Weighouttherequiredamountofagarose
(dependingonthegelpercentage)intoan
Erlenmeyerask.
2. Addtheappropriatevolumeofthebuffer
(30mMNaCl,2mMEDTA,pH7.5)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.
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.
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.
8. Immersethegelforatleastonehourintothe
alkalineelectrophoresisbuffer(30mMNaOH,
2mMEDTA).Dilute5volumesoftheDNA
sampleorladderwithonevolumeof6Xalkaline
electrophoresisloadingbuffer(180mMNaOH,
6mMEDTA,18%Ficoll400,0.05%bromcresol
green).
9. Heatsamplesandladderat70°Cfor5min,then
chillonicefor3minutes.Loadontothegel.
10. Runelectrophoresisat3V/cminalkaline
electrophoresisbuffer(30mMNaOH,2mMEDTA)
untilthedyerunsapproximatelytwo-thirdsofthe
waydownthegel.
Afterelectrophoresisthegelshouldbeimmersedfor30
minin100-300mlof0.5MTris-HClbuffer,pH7.5and
laterstainedina0.5µg/mlethidiumbromidesolutionfor
30min.Ifstainingisnotenough,thewholeprocedurecan
berepeated.
Warning.Hot agarose solution should be handled very
carefully.
North America
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Tel 800 235 9880
Fax 800 292 6088
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Alkaline Agarose Gel Electrophoresis
This protocol is for the Alkaline Agarose Gel Electrophoresis
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