Transformation

1. Totransform,mix0.1mlaliquotsofcellswithDNA(1-10ng).Leaveonicefor10-30min.

2. Heatshockat42Cfor2min(90-120sisOK)

3. Add0.9ml2XYTorLBandallowexpressionfor30-60minbeforeplating.

4. IfusingX-galandIPTG(optional),place0.1mlexpressionmixontheplate,add40ælX-galand4ælIPTG,andspreadtogether.Thisensuresthatwherevercellsgo,X-galdoestoo!.Betterstill,addIPTGandX-galtotheplate(0.5mlX-galand50mlIPTGper100mlagar).

---

Media,buffersandsolutions
5. YT,2x

6. LB

7. CaCl20.1M

8. MgCl2,0.1M

9. ice

10. glycerol

    ---

    ---

    CaCl2competentcells

    Contributor::Zappe,H

    Reference:DagertandEhrlich(1979)Gene6:23-28.

    ---

    1. Grow5mlE.coliin2XYT

    2. Dilute1mlculturein100mlfresh2XYT

    3. GrowtoOD600=0.2-0.4

    4. Centrifugationat5000rpmfor5minat4C

    5. Resuspendthecellsin50mlof0.1Mice-coldMgCl2.

    6. Holdonicefor30min

    7. Centrifugationat5000rpmfor5minat4C

    8. Resuspendin10mloficecold0.1MCaCl2.

    9. Mixandleaveonicefor30min,thenstore/use

    Transformation

    1. Totransform,mix0.1mlaliquotsofcellswithDNA(1-10ng).Leaveonicefor10-30min.

    2. Heatshockat42Cfor2min(90-120sisOK)

    3. Add0.9ml2XYTorLBandallowexpressionfor30-60minbeforeplating.

    4. IfusingX-galandIPTG(optional),place0.1mlexpressionmixontheplate,add40ælX-galand4ælIPTG,andspreadtogether.Thisensuresthatwherevercellsgo,X-galdoestoo!.Betterstill,addIPTGandX-galtotheplate(0.5mlX-galand50mlIPTGper100mlagar).

    ---

    Media,buffersandsolutionsForm
    5. YT,2x

    6. LB

    7. CaCl20.1M

    8. MgCl2,0.1M

    9. ice

    10. glycerol

        ---

        ---

        DMSOcompetentcells

        Contributor::Zappe,H

        Reference:ChungandMiller(1988)N.A.R.16:3580.

        ---

        1. Grow5mlovernightofthestrainin2XYTinastandardcontainer.Screwcaptightandlieflatontheshakerforgoodaeration.

        2. Diluteovernightculture1/100-1/200to25,50or100ml2XYTbrothinflasks5-10Xculturevolume(i.e.25mlin250mlflasketc.).

        3. Growtoearlylogphase(OD600=0.2-0.4)(90-180mindependingonthestrain)

        4. Collectcellsbycentrifugation(4000-5000rpmfor5minor5000xgfor5min)at4C.

        5. Cellsareresuspendedin1/10thculturevolumeoficecoldTSBandheldonicefor10minbeforetransformation.

        Transformation

        1. Fortransformation,0.1mlcellsaremixedwithupto0.1ægDNAandheldonicefor10min.

        2. Add0.9mlTSBGandincubate37Cfor30-60minbeforeplating.

        3. Competentcellscanbefrozenindry-ice/ethanolandstoredat-70C.

        ---

        Media,buffersandsolutionsForm
        4. YT,2x

        5. TSB,TSBG

        6. dry/iceethanol

           ---

           ---

           DMSOcompetentcells

           Contributor::Zappe,H

           Reference:ChungandMiller(1988)N.A.R.16:3580.

           ---

           1. 5mlovernightE.coliin2XYT

           2. Diluteovernightculture1/100-1/200to25,50or100ml2XYTbrothinflasks5-10Xculturevolume(i.e.25mlin250mlflasketc.).

           3. Growtoearlylogphase(OD600=0.2-0.4)(90-180mindependingonthestrain)

           4. Collectcellsbycentrifugation(4000-5000rpmfor5minor5000xgfor5min)at4C.

           5. Cellsareresuspendedin1/10thculturevolumeoficecoldTSBandheldonicefor10minbeforetransformation.

           Competentcellscanbefrozenindry-ice/ethanolandstoredat-70C.

           Transformation

           1. Fortransformation,0.1mlcellsaremixedwithupto0.1ægDNAandheldonicefor10min.

           2. Add0.9mlTSBGandincubate37Cfor30-60minbeforeplating.

           ---

           Media,buffersandsolutionsForm
           3. YT,2x

           4. TSB,TSBG

           5. dry/iceethanol

              ---

              ---

              DNAisolationfrombacterialcells

              Description:

              A.ThemethodofZappeisquitewordyandappliestomostbacteria

              protocol

              B.LargescalebacterialgenomicDNAprepbyCoyne

              protocol

              ---

              ---

              DNAisolationfrombacterialcells

              Contributor::Zappe,H

              Reference:BasedonthemethodofMarmur(J.Mol.Biol.3:208-218,1961).ModifiedbyJ.L.Johnson(VirginiaTech.)

              Thevolumesofbuffersgivenareforculturesof300-600mlofE.colitypecultures(goodgrowers).Reducevolumesforlowdensityorsmallercultures.Notethatmycobacteriamayhaveasimilarculturedensity,butDNAyieldwouldbemuchlowerduetothecellenvelope.

              1. Harvestthecellsbycentrifugationat7500RPMfor10min.Decantthesupernatantandallowthecentrifugationbottlestodrainupsidedownonpapertowel.Dothisunderappropriateconditionsforpathogens.Resuspendthecellsin20mlofsuspensionbufferforenzymaticlysisor10-20mlfordisruptionbyFrenchpressurecelltreatment.See2ciiforglassbeadmethod.Thelasttwomethodsrequireathinpasteofcells-nottoodilute.Resuspendthecellsproperly,eitherbyvortexingthepelletwith1or2mlbufferbeforeaddingtherestofthebuffer,orbydrawingupandexpellingthecell/suspensionbuffermixturewitha10mlPipette.Thereshouldbenoclumpsofcellsafterthisstep.Transferthecellstoasuitableflask(125-150ml).

              2. Digestthecellswithlyticenzyme.

              2a.Gram-negativebacteria.Adddrylysozyme(4mg/mlorabout1/8teaspoonfor25ml!)andincubate.Thetimeandtemperaturecanvaryfrom10minatroomtemperatureto4hat37Cdependingontheorganism.Removesmallsamples(100ml)periodicallyandmixwith100mllysingsolution.Ifthecellslyse,gotostep3,ifnotcontinueincubation.Morelysozymecanalsobeadded.

              2b.Gram-positivebacteria.Adddrylysozyme(8mg/mlorabout1/4teaspoonper25ml!)andincubateat37C.Startdoingthelysistestfrom15minonwards.Otherenzymesarealsoavailableiflysozymedoesnotwork,butlysozymeisthecheapest.

              2c.Recalcitrantbacteria.Someorganismswillnotbemadesensitivetodetergentlysisusinglyticenzymesandmustbephysicallydisrupted.Eitherofthefollowingmethodscanbeused.

              i.FrenchPressurecell.PassthecellsuspensionthroughtheFrenchPressurecellat16000psi(upperlimitformanycells)intoanequalvolumeoflysingsolution.

              ii.Glassbeads.UsingaBraunhomogeniserwithliquidCO2cooling.ScrapethecellpelletformthecentrifugationbottleandplaceitintoataredBraunshakerbottle.Makeuptheweightto20gwithsuspensionbuffer(cells+buffer),add20mlglassbeads(0.1mmdiameter)andshakefor5minat4000cyclesperminute(usethecoolingsystem).Separatethelysatefromtheglassbeadsbyfiltrationthroughacoarse60mlscinteredglassfilter.Use20mllysingsolutiontowashthelastofthelysatefromthebeads.

              1. Addanequalvolumeofthe2Xlysingsolution(unlessitsalreadybeenaddedinstep2cioriiabove)andavolumeof5MNaClO4equalto1/4ofthecombinedtotal.Thismixtureformsaprecipitate-heatto50Ctogetitbackintosolution.IfthecellsweresusceptIBLetolysis,mixwithaswirlingactiontogetauniformmixturebeforeallthecellslyse.Lysisisevidentbytheturbidsolutionbecomingtranslucentandveryviscous.Incubatethelysatefor4hat50-60Ctodegradecellularproteins.Aslittleas1hmaybeOK,butovernightissometimesconvenientandbetter.

              2. Add15mlphenol-chloroformsolution,shakebyhandtoformauniformemulsionandthenshakeonawristactionshakerfor20min.Transferthemixturetoa50mlcentrifugetubeandcentrifugeat12000RPMfor10minatroomtemperature.Meanwhilerinsetheflaskandletitdrain.Slowlydecantasmuchoftheupperaqueouslayerbackintotheflaskasyoucan.Donotallowanyoftheorganicphasetogoover.Theremainderoftheaqueousphasecanbecollectedbyusinganinverted2or5mlglasspipetteandapi-pump.DonotusenarrowborepipettesorGilsontipsassignificantshearingoftheDNAcanoccur.Repeattheextractionproceduretwicemore.Extractionofproteinsiscompleteifthereislittleornowhiteprecipitateattheinterfaceafterthecentrifugationstep.

              3. Carefullydecanttheaqueousphasefromthelastphenol-chloroformextractionintoasuitableflask(125mlErlenmeyer).Taringtheflaskandthenweighingthesolutionisagoodwayofestimatingthevolume(1ml=1g).Add0.6volumeofIsopropanolandswirltomix.Thenucleic(DNAandRNA)willprecipitateandformalooseclot(unlessthecellswerephysicallydisrupted,whereitwillbenecessarytocentrifugethesolution).HoldtheclotbackwithaPasteurpipetteandpouroffthelysate-Isopropanolsolution.Add25ml76%ethanolandallowtostandfor10min.Decanttheethanolandrepeatthewash.DecanttheethanolandpresstheclotofnucleicacidwithaPasteurpipettetogetridofmostoftheethanol.Allowtheprecipitatetodryat37Cfor15min.NevercentrifugepreparationsofhighMol.wt.DNA-theytakeaverylongtimetoresuspend(days).

              4. Dissolvethenucleicacidprecipitatein20mlTEbuffer(maytakeafewhours),add0.25mlofRNasemixandincubateat37Cfor1h(Notethatinanynucleicacidpreparation,mostofthenucleicacidatthispointisRNA).Extractthesolutiononcewith5mlchloroform:isoamylsolution,centrifugeandsavetheaqueouslayer.

              5. Add0.1volumesof3MSodiumacetate,mix,overlaywith2volumesof95%ethanol,andcollecttheDNAbyspoolingontoaglassrod(orPasteurpipette)byrotatingtherodandstirringthesolutionatthesametime.Continueuntilbothphasesarecompletelymixed.Alternatively,theDNAcanbeprecipitatedjustbyswirlingthesolutionasin5above.Collectthe"clot"ofDNA,washanddrytheprecipitateasin5above.

              6. DissolvetheDNAin3-5mlTE(again,thiscouldtakeaday-leaveat4C).Doa310-220nmscantodeterminetheactualconcentration.Storeataconcentrationofatleast0.5mg/mlat-20C.

              Note:TheprocedurecanbestoppedandtheDNAsolutionstoredat4Cforuptoafewdaysanyhwereafterstep4.However,donotstoresolutionscontainingphenol-decanttheaqueousphaseandstorethis.

              7. ---

              8. Media,buffersandsolutionsConcFormStorage

              9. Cellsuspensionbuffer1xliquidstock

              10. Lysingsolution2Xliquidstock

              11. NaClO45M

              12. Isopropanol

              13. Ethanol

              14. sodiumacetate3M

              15. TEbuffer

              16. Chloroform:Isoamylmix(24:1)

              17. Phenol-Chloroformmixture

              18. RNasemix

              20. Note:Ifthecellsaregoingtobephysicallydisrupted(Frenchpressofglassbeads),thendonotaddthesucrosetothecellsuspensionbuffer.

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