TailPreps:DNAIsolationFromMouseTailsWithoutPhenol

NOTE:THISISFINEFORSOUTHERNS,BUTNOTFORSCREENINGBYPCR.(fromRuixia,7/99,fromprotocolbyStefOehen7/94).

1.Put1cmtailin1.5mlmicrocentrifugetube.

2.Add500µlTailBufferand30µlProteinaseK.(seerecipesbelow).

3.IncubateO/Nat55°ree;C(or4hoursminimum).

4.Mixwellbyshakingbyrepeatedinversion.DoNOTvortex.

5.Letcooltoroomtemperature.

6.Pre-Spin(togetridoffur)8min(usually4°ree;,TRmok).

7.Poursupernatantintoacleantube(numbered).(Tossoutfurrytube).

8.Add250µlsaturatedNaCl(~6M)andinverttomix(don"tvortex)toprecipitatetheproteins.

9.Letsitfor5minutesatroomtemperature.

10.Centrifugemaxspeedinmicrofuge(~13000rpm),10minat4°ree;C,tospindowntheprotein.(TRmisok,but4°ree;ismuchbetter).

11.Pipetsupernatantintoafreshtube.Avoidtransferringthewhiteprecipitate.

12.Add500µlisopropanol.

13.MixbyinversionuntilawhitethreadymaterialisvisIBLe(DNA!).Shakeuntilthreaddoesn"tgetanybigger,~aminute.

14.Centrifugemaxspeedinmicrofuge(~13000rpm),10min(TRmisok).

15.Pipetoffthesup,beingcarefulnottodisturbthepellet(DNA).

16.Washpelletwith70%EtOH(100-800µl).Vortexbriefly.

17.Centrifuge6minutes.

18.Pipetoffthesup,beingcarefulnottodisturbthepellet(DNA).

19.Tap/Draintubeontoakimwipe.

20.AirDryfor45-60min.(DoNOTuseSpeed-Vac).

21.Dissovethepelletin100µlTris(10mMTris,oruseTE)containingRNase.

22.LetdissolveO/Nat4°ree;C.IMPORTANT-DON"TSKIPTHISSTEP!Mixwell.MeasureOD260orOD260/280.Use20µlforSoutherns,orcheck[DNA]anduse10mg.TailPrepSolutions(non-phenolprep.)RNase:10mg/ml.Use20µRNaseper1mlTris.ProteinaseK(Sigma#63173):10mg/mlinH2O,(activate1hrat37°ree;Cbeforeuse).SaturatedNaCl:AddNaCltoapproximately6M(35g/100ml).

TailBuffer

Stock	Reagent	Vol.For100mls	Final
1M	Tris	5mls	50mM
0.5M	EDTA	20mls	100mM
10%	SDS	5mls	0.5%
mQH2O	70mls

ShirleyReynolds

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