DNA Extraction from Frozen Tissue Sections

Tissuecollection,storage,microdissection,sectioning:Seeseparateprotocol.

Tissuehandling:NotethatallfreshtissueshouldbehandledasBioSafetyLevel2materials(weargloves,labcoat,etc.).

DNAextraction:ThefollowingprotocolisbasedonastandardphenolDNAextractionprotocol.Otherprotocols,andversionsofthisprotocol,arealsoacceptable.

1.Takepre-cutsamplesoutofthe–80°Cfreezerandthaw.

2.Add10mlofPBS(Ca-MgFree)inhoodinBSL2roomtodissolveOCT.Inverttubetomakesurealloftissueisinthesolutionandnotstuckonthetubewalls.

3.Spindowntissue1400rpm(500xg)for5min.

4.Removesupernatantcarefullywatchingthetissuepellet.

Note:Repeatsteps2-4(with5mlsPBS)ifitlookslikethereissignificant“sticky”OCTleftinthetube.IfyouarerepeatingthePBSwashstepyoudonothavetogettooclosetothetissuepelletthefirsttime.

5.Resuspendpelletbyvortexing.Add950uldigestionbuffer(100ul10XPCRbuffer[100mMTRIS,15mMMgCl2,500mMKCl),5ul0.5%tween20,845ulH2O)and30-50ulof20mg/mlProteinaseK(PK,SigmaP2308).

Note:thisvolumeshouldvarydependingonthesizeofthetissuepellet.Ifthepelletisbiggerthenadd2mltotalofbuffer+PK.

6.Resuspendpelletbyvortexing,andpipetingupanddownandplaceinashaking50°Cwaterbathovernight.Thenextdaymakesureallthetissuehasbeendigested.Ifnecessary,addmorePKbufferandallowtodigestforafewmorehours.

7.Spliteachtubeintotwo1.5mltubes(500ulpertube).

8.Add500ul(orequalvolume)ofroomtempPhenol/Chloroform/IsoamylAlcohol(PCIfromAmaresco)intoeachtubeandvortexfor10sec.

Note:PCIisthelowerorganiclayer.

9.Centrifugeat14000rpm(max)for2minatroomtemp.

10.RemoveaqueouslayerintoanewtubeandrepeatthePCIextraction(steps8-10).

11.Aliquottheaqueousphaseintoasfew1.5mlepindorftubesaspossible.Maximumvolumepertubeis350uls.Add1/2volumeof7.5Mammoniumacetateandmix.

12.Add2.5X100%ethanol,mixbyinversion.LeaveatRTfor2hrs,orONat–20°C.

13.Centrifugeat14000rpmfor15min@4°C.

14.Decantsupernatantimmediately.***Notewatchforthepellet***

15.Washpelletin70ulof100%ethanol.Makesureyourinsesides,rimoftube.Spinat14000rpmfor5minanddumpsupernatant.

16.BlotwithKimwipe.Airdrypellet.

17.Add20-50ulofTE.Thevolumewilldependonthepellet.(avg~30ul)

18.LeaveatRTfor2hrsorON.

19.Placetubesinto65°Cfor1hour(toinactivateDNAse).

20.Combinetubes.Rinseout“empty”tubeswith20ulTE(thesame20ulcanbeusedtorinseoutalltubes).

21.MeasureDNAconcentrationusingafluorometerwithaknownstandardDNAsolution.VerysmallamountsofDNAcanbequantitatedbyTaqMananalysiswithastandardassay.

22.StoreDNAat4°Cforshortperiods,orcolderforlongerperiods.RepeatedfreezingandthawingmayleadtoshearingofDNAintosmallerpieces.