PEGPreparationofPlasmidDNA

PlasmidDNAisolatedbythisprocedurecanbeusedroutinelyforelectrophoreticanalysis,restrictionendonucleasedigestionandtransformationofE.Coli.,DNAsequencing,PCRandmostothermolecularBIOLOGicaltechniques.TheprocedureisamodificationoftherapidalkalinelysismethodofIsh-HorowitzandBurke(1981).

Youwillneed3basicsolutions:-

SolutionI:50mMglucose,25mMTris.Cl,pH8,10mMEDTASolutionII:0.2MNaOH,1%(w/v)SDSSolutionIII:3Mpotassiumacetate,2Maceticacid,pH4.8.

1)Growovernightculturesofbacteriacontainingtheplasmidofinterest.

2)PelletbacteriabycentrifugationandresUSPendin0.02culturevolumesofSolutionI.

3)Addtothissuspension0.04culturevolumesofSolutionII.MixcontentsofthetubebyGENTLEinversionseveraltimes.

N.B.:Caremustbetakentomixtubecontentsgently.ThisavoidsmechanicalshearingofbacterialgenomicDNAwhichcouldcontaminatetheresultingplasmidpreparation.

4)Add0.03culturevolumesofSolutionIIIandinvert5-10timestomix.Shakethetubevigorouslyfor5seconds.AdditionofSolutionIIIcausesrenaturationofthegenomicDNAtoorapidlyandprecipitationoccurs.

5)Pelletbacterialdebrisbycentrifugationatroomtemperature(maximumspeedinamicrofugefor5minutesforsmallvolumes,10,000ginaMSEEuropaorequivalentfor20minutesforlargevolumes).

6)Removetheclearsupernatanttoacleantube.

7)AddDNase-freeRNaseA(0.01supernatantvolumeof10mgpermlstock),mixandincubateat37°Cfor30minutes.

8)Phenol/chloroformextracttoremovecontaminatingproteinsandprecipitatenucleicacidsbytheadditionofanequalvolumeofpropan-2-ol.

9)Pelletnucleicacidsbycentrifugationasdescribedabove(5).

10)WashtheDNAoncebrieflyin70%ethanolandairdryfor5minutes.

11)Re-dissolveDNAinwaterorTE(10ulpermloforiginalbacterialculture),addanequalvolumeof13%PEG8000,800mMNaCl,mixandincubateonicefor30minutes.

12)PelletpureDNAbycentrifugation(10minutesatroomtemperatureinamicrofugeor20minutesat10,000ginaMSEEuropaorequivalent),washoncein70%ethanolanddryundervacuum.

13)Dissolveinappropriatevolumeofsterile,nanopurewaterorTE,pH8.

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