AninexpensivealternativetoglassmilkforDNApurification

PreparationofSilica

1.SUSPend5gofsilica(Sigma,S-5631)in50mlofPBS.

2.Allowthesilicatosettlefor2h.

3.Discardthesupernatantcontainingfineparticulatematter.

4.Repeatsteps2and3.

5.Spinfor2minat2000g.Discardthesupernatant.

6.Add3MNaItomakeafinalconcentrationof100mgsilica/ml.

7.Storethesilicasuspensioninthedarkat4C.

RecoveryofDNAfromAgaroseGelUsingtheSilicaSuspension.

1.TotheagarosegelcontainingDNAfragments,addtwovolumesof6MNaI.

2.Incubatetheagarosein6MNaIat55Cfor5-10minwithoccasionalmixing.

3.Add10ulofthesilicasuspension.Vortexgently.Standfor5minatroomtemperaturewithoccasionalmixing.Onemgofthesilica(=10ulofthesilicasuspension)binds3-4.5ugofDNA.

4.Spinfor1mininamicrocentrifuge.Discardthesupernatant.

5.Pulsespin,andcarefullyremoveresidualliquid.

6.Suspendthepelletin500ulof50mMNaCl,10mMTrisHClpH7.5,2.5mMEDTA,50%(v/v)ethanol.

7.Spinfor1min.Discardthesupernatant.

8.Repeatsteps6and7.

9.Allowthepellettoair-dryfor10min.

10.Addanappropriatevolume(atleastonepelletvolume)ofTE.Vortexgentlytoresuspendthepellet,andstandfor5minatroomtemperaturewithperiodicagitation.

11.Spinfor1min.Transferthesupernatantintoanewmicrofugetube.

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