DNA试剂盒提取方法汉语版

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

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

3、s)2.Centrifugenomorethan3mlcultureor1×109cellsat4000×gfor10minutesatroomtemperature3.Aspiateanddiscardthemedia4.Add100μLTEbuffer.vortextocompleteresuspendthepellet5.Add10μLlysozyme6.Incubateat37℃for10minutesNote:theamountofenzymerequiredand/orthelengthofincubationmayneedtobemo

4、difieddependingonthebacterialstrainused.completedigestionofthecellwallisessentialforefficientlysis.longerincubationtimemayyieldbetterresult.Optional:followtheshortprotocolbelowfordifficulttolysebacteria.1.add25mgglassbeadsto1.5mlmicrocentrifugetube.2.Addsampletotheglassbeads3.

5、Vortexatmaximumspeedfor5minutes4.Letsamplestangtoallowthebeadstosettle5.Transfersupernatanttoanew1.5mlmicrocentrifugetube.7.Add100μLBTLbufferand20μLProteinaseKSolution.vortextomixthoroughly.8.incubateat55℃inashakingwaterbath.Note：usuallynomorethan1hourisrequiredforbacteriallys

6、is.ifashakingwaterbathisnotavailable,incubatethesamplesandshakeorbrieflyvortexevery20-30minutes.9.Add5μLRNaseA.INVERTTUBESEVERALTIMESTOMIX9.Incubateatroomtemperaturefor5minutes10.Centrifugeat10000×gfor2minutestopelletanyundigestedmaterial11.Transferthesupernatantoanew1.5mlmicr

7、ocentrifugetube.donotdisturbthepellet12.Add220μLBDLbuffer.votextomix13.Incubateat65℃for10minutesNote:awispyprecipitatemayfromuponadditionofBDLbuffer;itdoesnotinterferewithDNArecovery14.add220μL100%ethanol.vortexfor20secondsatmaximumspeedtomixthoroughly.Note：ifanyprecipitatecan

8、beseenatthispoint,breaktheprecipitatebypipettingupangdown10ti