1.TocellpelletinEppendorftube,add0.3g(roughly0.3ml)ofglassbeads,0.2mloflysisbufferand0.2mlofa1:1mixofphenolandchloroform.

2.Vortexthetubeattopspeedfor2min.

3.Add0.2mlofTE(10mMTris,1mMEDTA,pH8.0)andvortexagainforafewseconds.

4.Spinthetubesfor5min(roomtemperature)attopspeedinanEppendorfcentrifuge.

5.Transfertheaqueous(upper)phase(0.38ml)toafreshEppendorftube,usinganewPipettetipforeachsample.Discardthetubewiththeglassbeads.

6.Add2volumesof100%ethanolatroomtemperature.Mixthoroughly.

7.CentrifugeinEppendorffor2-3minatroomtemperature.

8.Discardthesupernatant(usetheaspirator;takecarenottodislodgethepellet).

9.Rinsethepelletwith0.5mlofcold,70%ethanoladdtheethanolslowlydownthesideofthetube,thencentrifugefor3-5sec.

10.Removethesupernatant.Leavethetubesopenandinvertedforthepelletstodry.(Ordrythepelletsundervacuum.)

Glassbeads

Use425-600micronbeads.SigmaG-9268workswellforus.Topreparethebeads:

• Pourthebeads(1kgbottle)intoa2litrebeaker.
• FillthebeakerwithconcentratedHCluntilthebeadsarefullysubmerged.Letstandinafumehoodfor15min.
• Washwithtap-distilledwateruntilthepHisneutral.Ithelpstorunthewaterthroughglasstubingjammedtothebottomofthebeakersothatthewaterflowsupfromthebottomofthebeaker.
• Transferthebeadstoabakingdishandbake(overnightisgood).
• Pourthebeadsintoasterilebottle.

Lysisbuffer:

10mMTris,pH8.0

1mMEDTA

100mMNaCl

1%SDS

2%TritonX-100