7/22/2017 PlasmidDNAExtractionfromE.

coliUsingAlkalineLysisMethodBIOPROTOCOL

MaterialsandReagents
1.RNAase(LifeTechnologies,Invitrogen)
2.Isopropanol(EMScience)
3.Ethanol(VWRChemical)
4.Tryptone
5.Yeastextract
6.NaCl
7.Glucose
8.EDTA
9.0.2NNaOH
10.SDS
11.KOAc
12.Potassiumacetate
13.Glacialacetate
14.TrisHCl(pH8.0)
15.LuriaBertanibroth(LB)medium:Bactotryptone(BDBiosciences),yeastextract(BDBiosciences)(seeRecipes)
16.Resuspensionsolution(P1buffer)(seeRecipes)
17.Lysissolution(P2buffer)(seeRecipes)
18.Neutralizingsolution(P3buffer)(seeRecipes)
19.TE(seeRecipes)

Equipment

1.Tabletopcentrifuges
2.1.5mleppendorftube
3.37Cheatblocker

Procedure

1.Growbacterial(E.coli)cultureinLBmediumwithappropriateantibioticsat37Covernight(O/N)withshaking.For
>10copiesplasmid,3mlcellcultureisusuallyenough.
2.TransferO/Nculturetoa1.5mleppendorftube,andspindowncellculture(twice)athighspeedfor1minattable
topcentrifuge.
3.Discardthesupernatant.Toremovetheliquidcompletelybyupsidedowntubeontoapieceofpapertowelforafew
sec.
4.Add100lofresuspensionsolution(P1buffer)intoeachtube,andvortextocompletelyresuspendcellpellet
5.Add100loflysissolution(P2buffer)andmixbygentlyinvertingthetube56times.Thesolutionshouldquickly
turntransparentandbecomemoreviscousindicatingbacterial
lysishastakenplace.
6.Add150lofneutralizingsolution(P3buffer)andmixbyinvertingthetubesseveraltimes.Atthispointbacterial
chromosomalDNAisusuallyseenasawhiteprecipitate.
7.Centrifugethetubesathighspeedfor10min.
8.Carefully transfer the supernatant (try to not disturb the white precipitate) to a new labeled 1.5ml eppendorf tube
witha1mlpipette.
9.Add 2.53 volume of 200proof cold ethanol (stores at 20 C) to each tube and mix by inverting the tubes a few
times.
10.SpindownplasmidDNAprecipitate(transparencypellet)athighspeedfor10min.
11.Discardthesupernatantandremovetheremainingliquidasmuchaspossiblebyleavingthetubeupsidedownona
pieceofpapertowel,thenkeepthetubesinatubeholderandairdryfor1020min.Todryfaster,keeptubesat37
Cheatblocker.DNAprecipitateturnswhitewhendry.
12.ResuspendtheDNApelletwith50lTE.Completelydissolvethepelletbypipettingsolutionseveraltimes.
Note:LargeamountsofRNAispresentintheDNAsample.Therefore,forsubsequentreactions,forexample,to

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7/22/2017 PlasmidDNAExtractionfromE.coliUsingAlkalineLysisMethodBIOPROTOCOL

digestplasmidDNA,add15l(1mgml1)RNAasetothedigestionsolutiontocompletelyremoveRNA.Or,add
RNAasedirectlytotheresuspensionsolutionwithafinalconcentrationof1mgml1.

Recipes

1.LBmedium
1%Tryptone
0.5%yeastextract
200mMNaCl
2.Resuspensionsolution(P1buffer)
50mMglucose
10mMEDTA
25mMTris(pH8.0)
Storeat40C.
3.Lysissolution(P2buffer)
0.2NNaOH
1%SDS
Storeatroomtemperature.
4.Neutralizingsolution(P3buffer)
3MKOAc(pH6.0)
For100mlsolution,60ml5Mpotassiumacetate(49.07gpotassiumacetatein100mlH2O)
11.5mlglacialacetateand28.5mlH2O,storeatroomtemperature.
5.TE
1mMEDTA
10mMTrisHCl(pH8.0)
Note:P1,P2,P3buffersfromtheQIAGENDNAextractionkitalsoworkwell.

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