DNAfromTailBiopsies

1.Remove0.5cmoftailintopolypropylenemicrofugetube(donotmince).(Thetubesmusthavetight-fittingcaps,sothattherearenoleaksinsteps3and7below.)

2.Add0.5mlDNAdigestionbufferwithproteinaseKaddedto0.5mg/mlfinalconcentration.(0.5mg/mlisahighconcentrationandcanprobablybereduced.)

DNAdigestionbuffer:50mMTris-HClpH8.0100mMEDTApH8.0100mMNaCl1%SDS

3.Incubateovernightat50-55°Cwithgentleshaking.(Atthisstep,mechanicalagitationgreatlyaidscompletedisruptionofthetail.)

4.Quickspintubestogetsolutionoffinsideofcap.

5.Fillinsidewellofmicrofugetubecapwithvacuumgrease.(WeuseDowCorninghighvacuumgreaseanda10ccsyringetodispense.)

6.Add0.7mlneutralizedphenol/chloroform/isoamylalcohol(25:24:1).

7.Mixfairlyvigorously.(DoNOTvortex--Weuseaclinicalrotatorfor1hour.)

8.Spininmicrofugeattopspeed5minutesandtransfer0.5mloftheupperphasetonewmicrofugetube.(UseP1000fortransfer,anddrawtheaqueousphasegentlythroughtipseveraltimesaftertransferiftheDNAisstillinlarge,gelatinousmass.)

9.Add1ml100%ethanolatroomtemperatureandinvert(usingclinicalrotatorifyouwish)untilDNAprecipitateforms.(approximately1minute)

10.Spininmicrofuge5minutesandcarefullyremoveanddiscardsupernatant.

11.Add0.5-1ml70%ethanol(-20°C)andinvertseveraltimes.

12.Spininmicrofuge5minutesandcarefullyremoveanddiscardsupernatant.

13.Quickspintubesandremovelastdropofethanolsolutionwith25µlcapillarytube.

14.Airdryatroomtemperatureorindessicator(overnightifyouwish).

15.Add100-200µlTEbufferandincubateat65°Cfor15minutestoresUSPendDNA.DrawDNAthroughP1000tipafter65°Cincubationtoaidinsuspensionifyouwish.

16.Use10-20µlforrestrictionenzymedigest.

17.Totalyieldisapproximately20-50µgDNA,0.1-0.25µg/µl.