SilicaClean-upofDNA

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Materials:

SilicaSUSPension:	add2gofsilicato15mlofH2O wash3xbycentifugationat2000xgfor2min estimatevolofsilicaandresuspendin2volH2O
SilicaWashSolution:	50mMNaCl,10mMTris7.5,2.5mMEDTA,50%Ethanol
6MNaI	note:lightsensitive

Procedure:

1. Add5volumesof6MNaItoDNAsolutionoragarosegelslice
   • ifgelslice,meltat55degreesfor5minuteswithoccasionalagitation
   • foraqueoussolutionsadd1/10vol3MNaOActoensurepHislessthan7
2. BindDNAtosilica
   • addappropriatevolumeofsilicasuspension(bindsapprox200ngofDNA/ulofsuspension)andmixwell
   • sedimentsilicamatrixbycentrifugation(10secondsin"nanofuge"orbriefspinatfullspendinmicrofuge)
   • washsilica3timeswith500ulwashsolution
   • pelletsilicaoncemoretoremoveanyresidualwashsolution
   • airdrytoremoveanyresidualEtOH
3. EluteDNAfromsilica
   • resuspendsilicapelletindesiredvolumeof10mMTris7.5orTEor1Xrestrictionbufferfordownstreamdigests
   • heat5minutesat60degrees
   • spin2min@fullspeedinmicrocentrifuge
   • removeDNA-containingsupernatanttocleantube

Notes&Misc:

• ConveinientPCRcleanupbeforedigestion:100ulPCRreactioncleanedupwith50ulsilicaandelutedfromsilicawith30ul1Xrestrictionbuffer
• ProcedureusefulforcleaningDNAfromPCRreactions,restrictiondigests,etc.aswellaspurifyingfromagarosegelslices
• If6MNaIsolutionbecomesyellow,itwillstillbindDNAif1/200volume10%aceticacidisaddedtolowerpH
• ProcedurealsousefulforconcentratingDNAfromdilutesolutions

References:

• AninexpensivealternativetoglassmilkforDNApurification.Boyle,J.S.andLew,A.M.(1995).TrendsinGenetics11(1):8
• GeneCleanProtocols.Bio101

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