DNA preparation from mouse tails

GeneralprecautionstobefollowedduringhandlingofanygenomicDNAsample:

AvoidShearingoftheDNAbylimitingmixingtoinversionandgentleshaking.Vortexingofsamplesisnotrequiredtoperformthefollowingprotocols.

AvoidcontaminationbyrigorouslycleaningpipetmanandusingautoclavedtipsduringmanipulationofgenomicDNAandreagentstobeusedwithgenomicDNA.

DNAPreparation:

1.Micearetoe-clippedandapproximately0.5-1cmoftheirtailissnippedoffandplacedintoa1.5-mlmicrocentrifugetube.Digestionisimprovedifthetailpieceiscutintoseveralsmallerpieces.Thistailcantheneitherbeprocessedimmediatelyorfrozenat-20¡C.Undernocircumstancesshouldtailsbeleftatroomtemperature.Ifthemiceareyoungerthan21days,theycanbetoe-taggedandthetailcutforanalysis.

2.Toeachtail,add600mloftaildigestionbuffer(whichhasbeensupplementedwithproteinaseKatafinalconcentrationof0.5mg/ml)andplaceat55¡Cfor6-24hours.OKtouseepindorfrepeater.

MixingduringthedigestionperiodwilldecreasethetimerequiredtodigestthetailsampleandimproveDNAquality.

Taildigestionbuffer(w/oproteinaseK):

• 315mlofautoclaveddeionizedwater
• 25mlof1MTris-HC1(pH8)
• 10mlof5MNaCl
• 10mlof0.5MEDTA(pH8)
• 50ml10%SDS(or10XSET)
• Priortouse,50mlofproteinaseK(10mg/ml)isaddedforeachmlofdigestbuffer

3.Oncethetailmaterialisdigested,eachsampleisextractedoncewith600µlphenol:chloroform(75:25)(addwithrepeater,inverttubesmultipletimestomixorvortex).Centrifugethesamplesat13,000RPM(max)foratleast5min.toseparatetheorganicandaqueousphases,theaqueous(top)phaseistransferredtoanewmacrocentrifugetube(useawideborePipettetip).Donottrytotransfertheentireaqueousphasetoanewtube.Donottransferanyphenol/chloroform.

4.Extracttheaqueoussamplewithchloroform(600µl,mixwellasnotedabove),butthistimeinsteadoftransferringtheaqueousphasetoanewtube,removetheorganiclayerfromundertheDNAsample.ItisokaytoleavesmalldropletsofCHCl3.

5.Add1mlof100%ethanol(-20¡C)andmixthetubesbyinvertingthemseveraltimes(Makesuretubesareverywellmixed).TheDNAshouldformavisIBLemass.PellettheDNAbyspinningatmaximumspeedfortwominutes.Carefullypourofftheethanolandwashwith70%ethanol/0.1MNaCl(-20¡C).Mixbriefly,thencentrifugeatmaxRPMfortwominutes.Carefullypouroffthewashandinvertedtodryforseveralminutes.(WatcheachpelletanddonotletanyDNAslideoutofthetube!!!).Dependinguponthepelletsize,theDNAshouldthenberesUSPendedin50-200mlofTE.AllowtheDNAtodissolveovernightat4¡C.

ThisDNAisnowreadytobeusedinPCRorsouthernblotanalysis.