IsolationOfCosmid,PlasmidandP1DNAsviaDiatomaceousEarth-basedMaxi,MidiandMini-preps,*and*thenon-DiatomaceousEarth-basedMini-prep.

I.DiatomaceousEarth-basedMaxi-prep

A.Materials:

1.LB(LuriaBertani)media

10gBactoTryptone

5gBactoYeastextract

10gNaCl

Dissolvetheabovecomponentsindoubledistilledwater.Adjustthevolumetoaliterandautoclaveat121oC/15lbsfor20mins.Coolthesolutionandaddtheappropriateamountofantibioticjustbeforeuse.TypicallyAmpat25ug/mlorKanat50ug/ml.

2.TB(TerrificBroth)media

12gBactotryptone

24gBactoYeastextract

4mlGlycerol

Dissolvetheabovecomponentsinapproximately900mldoubledistilledwater.Autoclavethissolutionfor20minsat121oCat15psi.Letthesolutioncool,add100mlofTBSaltSolution(describedbelow),andadjustthefinalvolumeto1000mlbyaddingdoubledistilledwater.Addantibiotic,typicallyAmpat25ug/mlorKanat50ug/mljustpriorinnoculationwithcells.

10XTBSaltSolution(Fortotalvolume100ml)

0.17MKH2PO4=2.31gKH2PO4

0.72MK2HPO4=12.54gK2HPO4

Adjustthevolumeto100mlwithdoubledistilledwaterandautoclave.

3)GET/LYSOZYMEsolution(fortotalvolume100ml):

50mMGlucose=0.9gd-Glucose

10mMEDTA,pH8.0=1mlof1Mstock

25mMTris-HCl,pH8.0=2.5mlof1Mstock

Makethissolutionfreshorfiltersterilizebeforestoringat4oC.andadd2mg/mlLysozyme(0.2gmslysozyme/100ml1xGET)justbeforeuse.

4)NaOH/SDS(Alkalinelysissolution):

Makethissolutionfresh.Fortotalvolume100ml:

0.2NNaOH=20mlof1Nstock

1%SDS=5mlof20%SDS<

Makethevolumeto100mlwithdoubledistilledwater.

5)3.0MSodiumAcetate(NaOAc),pH4.86)DNAbindingmatrix

Weigh10gofdiatomaceousearth(SigmaD-5384)andresUSPenditin100mlofdoubledistilledwaterina100mlmeasuringcylinderandletitsettledownfor3hours.Decantthesupernatant.Ifyouwishtostorethede-finedmatrix,resuspendthepelletinTE:10:1,transferthematrixtoascrewcapstoragebottleandstoreinthecoldroom.Otherwise,resuspendthepelletin100mlof6Mguanidinehydrochloridecontaining50mMTris-HCland20mMEDTA(finalmatrixconcentrationof100mg/mlassumingnosignificantlossesduringthedefiningstep).

7)WashBuffer(TE/ethanol)Fortotalvolume1000ml:

10mMTris-HCl,pH8.0=10mlof1Mstock

1mMEDTA,pH8.0=1mlof1Mstock

50%Ethanol=500mlofabsolutealcohol

Makethevolumeto1000mlwithdoubledistilledwater.

8)ElutionBuffer(TE:10:1)-NotethatthisisTris-EDTA:10:1nottheusual10:0.1molarratioofTris-EDTA.

Fortotalvolume1000ml:

10mMTris-HCl,pH8.0=10mlof1Mstock

1mMEDTA,pH8.0=1mlof1Mstock

Makethevolumeto1000mlwithdoubledistilledwater.

9)EthanolAcetate

95%Ethanol=95mlofabsoluteethanol

0.12MNaOAc=4mlof3MNaOAc,pH4.8.

Makethevolumeto100mlwithdoubledistilledwater.

10)Isopropanol

11)Acetone

12)70%Ethanol

13)a)DNasefreeRNaseA

For100mlofRNaseAbuffer:

0.1MNaacetate=0.82ganhydrous

0.3mMEDTA=0.3mlof100mMstock

AdjustpHto4.8withaceticacid.Makethevolumeto100mlwithdoubledistilledwater.Aliquotin2mlamountsandstoreat-20oC.Add40mgsofpancreaticRNaseAtothe2mlalaquotsofRNAseAbufferjustbeforeuseandinactivateanycontaminatingDNasebyheatingat80oCfor10mins.

b)RNaseT1(10units/ul)madein50mMTris-HClpH7.6.

B.Methods:

1. Grow2litersofcellscontainingdesiredplasmid,cosmid,P1chimericvectorsinthefollowingmanner:
   • Inoculatecellsinto3mlofLBmediacontainingtheappropriateantibioticandgrowfor8hoursat37oCwithshaking.
   • Pourtheabove3mlcultureafter8hoursinto50mlofLBmediacontainingtheappropriateantibioticandgrowfor8hoursat37oCwithshaking.
   • Splittheabove50mlcultureintotwoaliquots.Pour25mlaliquotseachintoa2literflaskcontaining1literofLBmediawithappropriateantibiotic.Growthetwolitermedia(twoflasks)at37oCfor8hourswithshaking.

   • Pelletthecellsfromtheabovetwoliterculturebycentrifugationat5Kfor10mins.Cellsmaybefrozenatthispoint.

   • Letthecellsthawatroomtemperature.Resuspendthecellsin35mlofGET/LYSOZYMEsolutionwiththehelpofaspatula.(Caution:DonotvortexatanytimeduringtheprocedurebecauseyoumayshearthechromosomalaswellasthecosmidDNA).

   • Add70mlofALKALINELYSISsolution.Mixitgentlywithaspatulaandincubateonicefor5mins.Thesolutionbecomesveryviscousandstringy.

   • Add52.5mlof3MNaOAc,pH4.8.Mixgentlywithaspatulaandincubateonicefor60mins.

   • RemoveprecipitatedSDS,proteinandchromosomalDNAbyfilteringthroughadoublelayeredcheeseclothandcentrifugationat12Kfor10minsat4oC(twiceifrequired).

   • TotheclearsupernatantaddDNasefreeRNaseA(finalconcentrationofRNaseAintheclearedlysate~40ug/ml)andRNaseT1(40units/ml).Incubateat37oCfor30mins.

   • AddanequalvolumeofISOPROPANOLtotheclearRNasetreatedlysate.IncubateatR.Tfor5minsandspintheDNApelletdown.

   • Resuspendthepelletin1xTE(20-30ml=X).Add2Xvolumeof100mg/mlmatrix.AllowtheDNAtobindfor5mins.SpintheDNAboundmatrixdown.

   • Decantthesupernatantandwashthematrixpelletwith2XvolumeofWASHBUFFER.

   • Decantthesupernatantandwashthepelletwith2XvolumeofACETONE.

   • Decantthesupernatantanddrythepelletinthevacuumovenand/orat65oCuntildry.

   • ElutetheDNAfromthedriedpelletwith1XvolumeofELUTIONBUFFERat65oCfor10minswithintermittentstirring.

   • Spinthepelletdownbycentrifugationat12Kfor10minsandcollectthesupernatant.

   • Repeatsteps13)and14)forincreasedyieldofplasmid,cosmid,orP1vector.

   • Combinethesupernatantsandadd2.5volumesofETHANOLACETATEtoprecipitatetheDNA.

   • WashtheprecipitatedDNAwith1Xvolumeof70%ETHANOLanddrythepelletundervacuum.

   • ResuspendtheDNApelletin2mlofELUTIONBUFFER.Note:
     • Typicalaverageyield:
     • P1orCosmid:0.2-2ugpermlculture.
     • Plasmid(pUCorrelatedclones):2-4ugpermlculture.

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