DNA复制时辨认复制起始点主要靠 A.拓扑异构酶B.引物酶C.解螺旋酶D...
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- 3gnodules(freshorfrozeninliquidN2)weregroundtoapowderinmortarandpestlewithliquidN2.Tothepowderwasaddedicecold0.5Mmannitol(orsucrose),0.05MTris(pH7.5),0.02MNasuccinate,5mMNadithionite.PasstheresUSPensionthroughonelayerMiraclothandrinsetheclothwith5mlofthesamebuffersolution.Spinfiltrateinasterilecentrifugetubeandspin5min5minat1500rpm.Respinsupernatant5minat9000rpm.ResuspendpelletinLiClbuffercontaining10mMvanadylribonucleosidesandtreatasstatedabove.
0.1MLiCl,1%SDS,7Murea,0.1MTris(pH8.4),5mMEDTA
Extractwithphenol:chloroform3times.Precipitatewith2volethanol.Spin.Redissolveppt.in2mlH2Otreatedwith0.1%DEPCandautoclaved.Add6ml4MNaacetate(6)treatedwithDEPC.Incubateonice2hr,spin30minat8000rpm,andwashppt3timeswithicecold3MNaacetate(6)treatedwithDEPC.RedissolveRNAinDEPC-treatedH2O,ppt.with2.5volethanol,andstoreasppt.at-80C.
Cellsfrom100mlcultureinearlylogphaseGYPCwerespunandresuspendedin1mlsterile25%sucrose.Thissolutionwastransferredtoasterilecentrifugetubeand6.5mlM-STET,0.5mllysozyme(10mg/ml),and0.4ml200mMvanadylribonucleodideswereadded.
M-STET:4%sucrose,6%tritonX100,0.06MTris(pH8),0.06MEDTA
Incubateonice5min.Boil2min[oraddSDS/sarkosyl,extractwithphenol:chloroform,andppt.],chill,andspin10minat8000rpm.Tothesupernatantadd0.7volisopropanol,chill,spinagain,andresuspendpelletin5ml.
5XRunningbufferforRNA(formaldehyde)gels:(For500ml)2ml0.5MEDTA,6.8gNaacetate,12.8gMOPS(freeacid),9.0gMOPS(base).
- Inducecellsnormally-10mlof0.2OD600.
Washcells(grownO/NinORSminimalplusyeastat30or37C)2timeswithnifmediaanddilutingto0.2OD(10mlseach).Check1bottleforinductionbyacetylenereduction.
- Afterinductionadd0.5mlvanadylribonucleosidesdirectlythroughseptumintovialwithasyringe(vanadylprotectscellsagainstoxygen).
- Putcellsinplastictubes,spinafor2minat8000rpminSorvall.
- Resuspendin10MurealysisbufferwithplasticpasteurPipette.
- Add0.5-0.75mlofphenol:chloroform(50:50)["phenol"isphenol:m-cresol:hydroxyquinoline]tomicrofugetube.
- Vortex,placeat65Cfor2-3min.repeat.vortexandspin2-3mininmicrofuge.
- Re-extractliquidphase2moretimesatroomtempwithphenol:chloroform.Avoidproteinaceousinterphasewhenremovingupperliquidphase.
- Extractwithchloroform1time.
- Precipitatewith100%ethanol,0.3MNaacetate(5.2)at-20CO/N.
- Collectppt.,wash1timewith70%ethanol,1timewith100%ethanol,andresuspendin10mMNaacetate(5.2)-treatforgels.
formaldehydegels:
seemanual
runincoldroomO/Nat15Vinminigel;unit
blotdirectlytonylon-66membranes
deionizingformamide-usecolumns,donotaddresindirectlytoformamide.
- Blot15-45minwith50mMNaOH(autoclaved).
- Blot3-5hrswith45mMNaacetate(5.2).
- Exposetouvlamp2min.
- prehybridizewith10mg/mlheparin5min-4hr.
- hybridizeO/Nat42C.
- Wash:500ml5XSSPE30-45minat42C(46-54setting)500ml1XSSPE0.1%SDSat42Cfor45-60min.500ml0.1XSSPE0.1%SDSroomtemp.
Rewash1XSSPE
- Blotdry(nottotallydry!-donotairdry)putinsaranwrap.
- AutorADIograph.
Tostrip:
- Washat65C1-2hrsin1/10lysisbuffer.
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