Thermo Fisher Scientific Alkaline Agarose Gel Electrophoresis User guide

Brand
Thermo Fisher Scientific
Model
Alkaline Agarose Gel Electrophoresis
Type
User guide
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
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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Alkaline Agarose Gel Electrophoresis
This protocol is for the Alkaline Agarose Gel Electrophoresis
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Thermo Fisher Scientific Alkaline Agarose Gel Electrophoresis User guide

Brand
Thermo Fisher Scientific
Model
Alkaline Agarose Gel Electrophoresis
Type
User guide
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