InjectionDNAPreparation

1)Culture400mlsofbacteriaharboringtransposonovernightat37oC.

2)Harvestcellsat7000xgfor5min.(pelletcanbefrozenat-80oCforseveralweeks)

3)ResUSPendpelletthoroughlyin14mls:

for32mlsfor64mlsfor128mlsstocksoln50mMglucose1.6mls3.2mls1M25mMTris8.00.8mls1.6mls1MpH8.010mMEDTA0.64mls1.28mls0.5M

5)Add:

for32mlsfor64mlsfor128mlsstocksoln0.2NNaOH1.28mls2.56mls10N1%SDS3.2mls6.4mls20%

6)Add16mlscold3MKOAcpH4.8(instructionsformakingthisareinAppendixBoftheColdSpringHarborcloningmanual).Mixgently.Incubate30min.onice.(Longerisokay)

7)Spinat7000xgand4oCfor10min.Recoversupernatantthroughafunnellinedwith2layersofcheeseclothandintoa100mlgraduatedcylinder.Add0.6volumesofisopropanol.

8)Centrifugeat7000xgfor5-10min.Drain.Airdrypellet(butnotcompletely).

9)Resuspendpelletin3mlsTE.Adjustvolumeto4mlswithTE.Transfertoa5mlclearultracentrifugetube.

10)Add3.8gCsCl.CovertubewithParafilmandshakeuntilCsClisdissolved.Add0.4mlsof10mgs/mlEtBr.BalancetubeswithTEorsaturatedCsClsoln.

11)Centrifugeat40Kfor48hrsinanSW-55(orcomparable)rotor.InjectionDNAPreparation(cont)12)Pulllower(plasmid)bandusinga22.5gaugeneedleanda1mlsyringe.

13)ExtractEtBrwithwatersaturatedbutanolorwatersaturatedisopropanol.

13)Splitsampleinto2(forsafety)anddialyzevs.4-5changesofTEover~24hrs(dialysisbuffervolumeshouldbeatleast100xsamplevolume).

14)RecoverDNAfromdialysistubing,add1/4volume10MNH4Ac,and21/2volumeabsoluteEtOH.Spinat10,000xgfor10min.Rinsepelletwith70%EtOH.Drainpelletandairdry(butnotcompletely).ResuspendDNAinTE,determineconcentrationbyA260-280andstoreat4oC.

15)Justpriortoinjection,mix20ugoftransposonDNAwith20ugpWCDNA,andEtOHpptusing1/4volume1/4volume10MNH4Ac,and21/2volumeabsoluteEtOH.Rinsepelletwith70%EtOH.Drainpelletandairdry(butnotcompletely).ResuspendDNAin45ulofinjectionbufferand5ulof(filtered)greenfoodcoloring.

Comments:TheCsClbandingprotocolcanbescaledupifnecessary.Ioftenpool2-3litersworthofDNAinto110mlultracentrifugetube.

TheDNA(especiallyforlargerconstructs)ismuchnicerifitisnotdriedcompletelyafteranyofthealcoholpptsteps.