DNA试剂盒提取方法汉语版.doc

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1、.Thismethodallowsgenomicbacterialisolationfromupto3mlLBcultureMaterialsandequipmenttobesuppliedbyuser:TabletopmicrocentrifugeNuclease-free1.5mlmicrocentrifugetubesWaterbathcapableof37℃Shakingwaterbathcapableof55℃Incubatororwaterbathcapableof65℃100%ethanolIsopr

2、opanolTEbufferVortexerBeforestartingPrepareDNAwashbuffer.HBCbufferandlysozymeasinstructedinthe“preparingreagents”sectiononpage4Setanincubaororwaterbathto65℃Setawaterbathto37℃Setashakingwaterbathto55℃Heatelutionbufferto65℃1.culturebacteriainLBmediatolog-phase.(

3、overnightculturecanbeusedinmanycases)2.Centrifugenomorethan3mlcultureor1×109cellsat4000×gfor10minutesatroomtemperature3.Aspiateanddiscardthemedia4.Add100μLTEbuffer.vortextocompleteresuspendthepellet5.Add10μLlysozyme6.Incubateat37℃for10minutes..Note:theamountof

4、enzymerequiredand/orthelengthofincubationmayneedtobemodifieddependingonthebacterialstrainused.completedigestionofthecellwallisessentialforefficientlysis.longerincubationtimemayyieldbetterresult.Optional:followtheshortprotocolbelowfordifficulttolysebacteria.1.a

5、dd25mgglassbeadsto1.5mlmicrocentrifugetube.2.Addsampletotheglassbeads3.Vortexatmaximumspeedfor5minutes4.Letsamplestangtoallowthebeadstosettle5.Transfersupernatanttoanew1.5mlmicrocentrifugetube.7.Add100μLBTLbufferand20μLProteinaseKSolution.vortextomixthoroughly

6、.8.incubateat55℃inashakingwaterbath.Note：usuallynomorethan1hourisrequiredforbacteriallysis.ifashakingwaterbathisnotavailable,incubatethesamplesandshakeorbrieflyvortexevery20-30minutes.9.Add5μLRNaseA.INVERTTUBESEVERALTIMESTOMIX10.Incubateatroomtemperaturefor5mi

7、nutes11.Centrifugeat10000×gfor2minutestopelletanyundigestedmaterial12.Transferthesupernatantoanew1.5mlmicrocentrifugetube.donotdisturbthepellet13.Add220μLBDLbuffer.votextomix14.Incubateat65℃for10minutesNote:awispyprecipitatemayfromuponadditionofBDLbuffer;itdoe

8、snotinterferewithDNArecovery15.add220μL100%ethanol.vortexfor20secondsatmaximumspeedtomixthoroughly.Note：ifanyprecipitatecanbeseenatthispoint,breaktheprecipitatebypipettingupangdown