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DNAExtractionProtocol

ModifiedfromMoBio96WellManualExtractionMethod

Itemsnotprovidedintheextractionkit:

1. DNAdecontaminatingsolution(DNAaway,10%bleach,etc.)
2. Largevolume8channelpipette+tips
3. 100%Moleculargradeethanol
4. Sterilereagentreservoirs
5. Waterbathsetto65C
6. Icebath
7. Swingbucketcentrifugecapableof4500xg
8. Plateshakerwith4metalplateadapters.Thisprotocolusesthe96wellplateshaker(catalog
#11996)listedontheMoBiowebsite.

Itemsincludedintheextractionkit(for196wellextraction):

1. (1)Beadplate
2. (1)Spinplate(filter)
3. (1)0.5mlcollection
4. (4)1.0mlcollectionplates
5. (2)2.0mlcollectionplates
6. (1)Microplate(DNAelution)
7. SealingTape
8. CentrifugeTape
9. ElutionSealingMat

10.Labeledsolutions

Beforeyoustart:

1. CleanallsurfacesandpipettorstoremoveDNA
2. LabelPlates
1. Plate#11mlcollectionplate
2. Plate#21mlcollectionplate
3. Plate#31mlcollectionplate
4. Plate#41mlcollectionplate
5. Plate#52mlcollectionplate
6. Plate#62mlcollectionplate
7. UVSterilizeandLabelreservoirs
1. Beadsolution750ul/well
2. C160ul/well
3. C2250ul/well
4. C3200ul/well
5. C4650ul/well
6. C5500ul/well
7. C6100ul/well

Pleasewearglovesatalltimes.
1.BEFORETHEFIRSTUSEONLY,SolutionC5Dmustbeprepared.Addanequalamountof
100%EthanoltoSolutionC5D(forthe4prepkit=120ml,orforthe12prepkit=360ml).Mix
well.Putacheckmarkintheethanoladdedboxonthebottlecaplabel.

2.CentrifugeBeadPlatefor1minat2500xgtopelletthebeads.RemovetheSquareWellMatfromthe
PowerSoilhtpBeadPlateandsetaside.Add0.1to0.25gramsofsoilsampleorsampleswab.

Note:ThisisanappropriatestoppingpointandyoucanstorethePowerSoilhtpBeadPlateat4C
coveredwiththeSquareWellMat.Thisisthemosttimeconsumingstepoftheprotocol.Caremustbe
takentoavoidcrosscontaminationbetweensamplewells.

3.Add750lofPowerSoilhtpBeadSolutiontothewellsofthePowerSoilhtpBeadPlate.

4.CheckSolutionC1.IfSolutionC1hasprecipitated,heatsolutionat60Cuntiltheprecipitatehas
dissolved.

Note:SolutionC1containsSDS.Ifitgetscold,itwillprecipitate.Heatingat60CwilldissolvetheSDS.
SolutionC1canbeusedwhileitisstillwarm.

5.Add60lofSolutionC1.SecuretheSquareWellMat(fromstep2)tightlytotheplate.Placesealed
platesin65Cwaterbathfor10min.DONOTSUBMERGETHEPLATES.

6.PlacePowerSoilhtpBeadPlatebetweenthealuminumplateadaptersandsecurelyfastentothe96
WellPlateShaker.

7.Shakeatspeed20for20minutes.

8.Centrifugeatroomtemperaturefor6minutesat4500xg.

a.Whilecentrifuging,aliquot250lofSolutionC2intoeachwellofPlate#1andcoverwithSealingTape.
TheSealingTapecanbereusedwhencentrifugingPlate#1instep11ifhandledcarefully.

9.RemoveanddiscardtheSquareWellMatfromtheBeadPlate.

10.CarefullyremovetheSealingTapefromPlate#1andtransferthesupernatant(~400500l)fromthe
BeadPlatetoPlate#1andpipetteupanddown4times.

Note:Thesupernatantmaystillcontainsomeparticles.

11.ReapplytheSealingTapetoPlate#1.Incubateat4Cfor10minutes.CentrifugePlate#1atroom
temperaturefor6minutesat4500xg.

a.Whilecentrifuging,aliquot200lSolutionC3intoeachwellofPlate#3,thencoverwithSealingTape.
TheSealingTapecanbereusedwhencentrifugingPlate#3instep13ifhandledcarefully.

12.Aftercentrifugation,carefullyremoveanddiscardSealingTapefromPlate#1.

13.Avoidingthepellet,transfertheentirevolume(~600ldependingonsampletype)ofsupernatantin
Plate#1toPlate#2.
14.ApplySealingTapetoPlate#2andcentrifugeatroomtemperaturefor6minutesat4500xg.

15.CarefullyremoveSealingTapefromPlate#2andPlate#3.Avoidingthepellet,transfertheentire
volumeofsupernatant(~600l)fromPlate#2toPlate#3andpipetteupanddown4times.

16.ReapplySealingTapetoPlate#3.Incubateat4Cfor10minutes.Centrifugeatroomtemperaturefor
6minutesat4500xg.

17.CarefullyremoveanddiscardSealingTapefromPlate#3.Avoidingthepellet,transfertheentire
volumeofsupernatant(~750l)toPlate#4.

18.ApplySealingTapetoPlate#4andcentrifugeatroomtemperaturefor6minutesat4500xg.

a.Whilecentrifuging,add650lofSolutionC4toPlate#5.

19.Avoidinganyresidualpellet,transferupto650lofsupernatantinPlate#4toPlate#5.

20.Addasecond650l(1300lC4total)aliquotofSolutionC4toeachwellofPlate#5.

Note:ItissafetostoptheprotocolatthisstepandstorethesamplescoveredwithSealingTapeat4C.
Makesuretobrieflycentrifugetheplatetocollectanycondensateontheplatesealafterovernightstorage.

21.Pipetsamplesupanddowntomix.

22.PlaceSpinPlateontoPlate#6.

23.Loadapproximately650lfromPlate#5intoeachwelloftheSpinPlateandapplyCentrifugeTape.

24.Centrifugeatroomtemperaturefor5minutesat4500xg.DiscardtheflowthroughandplacetheSpin
PlatebackonPlate#6.CarefullyremoveanddiscardtheCentrifugeTape.

25.Repeatsteps2324untilallthesupernatanthasbeenprocessed.Discardthefinalflowthrough.

26.PlacetheSpinPlatebackonPlate#6.

27.ConfirmthatethanolhasbeenaddedtoSolutionC5D(seestep1).Add500lofSolutionC5Dto
eachwelloftheSpinPlate.ApplyCentrifugeTapetotheSpinPlate.

28.Centrifugeatroomtemperaturefor5minutesat4500xg.DiscardtheflowthroughandplacetheSpin
PlatebackonPlate#6.

29.Centrifugeagainatroomtemperaturefor6minutesat4500xg.Discardtheflowthrough.

30.CarefullyplacetheSpinPlateontotheMicroplate.RemoveCentrifugeTapefromtheSpinPlateand
discard.

31.Add100lofSolutionC6tothecenterofeachwelloftheSpinPlate.ApplyCentrifugeTape.LetC6
sitonthefilterfor10minutesatroomtemperaturebeforefinalcentrifugationstep.

32.Centrifugeatroomtemperaturefor7minutesat4500xg.RemoveCentrifugeTapeanddiscard.
33.CoverwellsofMicroplatewiththeElutionSealingMatprovided.DNAisnowreadyforany
downstreamapplication.Nofurtherstepsarerequired.

Prolongedstorageat4CwillresultintheevaporationofelutedDNA.WerecommendstoringDNAfrozen
(20Cor80C).SolutionC6doesnotcontainEDTA.ToconcentratetheDNAseetheHintsand
TroubleshootingGuideprovidedintheMoBioprotocol.

ImportantConsiderations

1.Normaldiameter1mlpipettipsaretoolargeforsomeofthepitettingsteps.Togetaroundthisproblem
weuseaRainin300ul8channelwithfilteredtips(Rainin#SRL300F)andaRainin1000ul8channel
withextendedlengthfilteredtips(Rainin#RTSL1000XF).

2.Makesurethatallofthenecessaryconsumablesandreagentsareinplacebeforeyoustarttheextraction.
Remember,eachpipettingstepwillrequire1boxof96tipsperplate.

3.WeuseindividuallywrappedreagentreservoirsandexposethemtoUVlightfor30minutespriorto
usage.

4.InStep7,itisimportantthattheplatenotrubagainstanysurfacesintheshaker.

5.Makesurethatthealphanumericgridisinthesameorientationacrossalltheplates.Onafew
occasionswehavenoticedthatthestickerisnotalwaysinthesamepositionontheplate.