请使用支持JavaScript的浏览器! KCF系列快开式静密封磁力搅拌反应釜结构及使用简介 jz.docin.com..._蚂蚁淘，【正品极速】生物医学科研用品轻松购|ebiomall -蚂蚁淘商城
欢迎来到蚂蚁淘！（生命科学科研用品平行进口B2B商城） 请登录 免费注册 询价篮
我的蚂蚁淘
产品分类
网站导航
微信关注
联系QQ
0
热门关键词 jackson二抗 Polyscience 23966 Liposomes脂质体 Biotium溴化乙锭 goldbio荧光素
我的购物车0
全部分类
酶
首页 品牌展示 产品查询 新闻资讯 帮助中心 联系我们 现货平台
商品列表 技术文章 相关问答
当前位置： > 首页 > 技术文章 >
KCF系列快开式静密封磁力搅拌反应釜结构及使用简介 jz.docin.com...
来自 : 蚂蚁淘

KoshlandLab,CarnegieInstitutehttp://www.ciwemb.edu/labs/koshland/Protocols/MICROTUBULE/mmb.html
  1. DeterminetheOD600andcorrelatethecelldensityfromthechart.Setupfour100mLYPDculturesatthefollowingdensities:0.7x105,1x105,and3.0x105cells/mL
  2. GROWTHOFCELLS
  3. Grow100mLofcellstoOD600=0.7-0.8at23oC.
  4. Add5-10ulofBME,15minutesbeforespin.
  5. Harvestcellsintwo50mLconicaltubes,spin1.5-2.0Kfor8-10min.
  6. SPHEROPLASTING.ResUSPendcellsin4mLYWB.*NOTE:Ifcellshavebeenarrestedwithnocodazoleoralphafactor,includetheinhibitorintheYWB.Letsitatroomtemperaturefor2-3min.Addoxalyticase[12uL1mg/mlstock].Incubateat23oCwithgentleshakingtoformspheroplasts.Spheroplastingshouldbecompletewithinonehour;avoidincubationslongerthan1h,15min
  7. TransferspheroplastedcellstoaTOMYtube.Spinat3.5Kfor7min.Resuspendcellsin4mLYWB(containing5%glyceroland1mMPMSF[0.2Mstockin100%EtOH])bygentlypipettingupanddown,usinga1mLpipet.Spinat3.5Kfor7min.
  8. Gentlyresuspendcellsin2.5mL1xEBB(containing5%glyceroland0.4mMPMSF).
  9. Allowcellsto"swell"for10min.atroomtemperature,thentransfertohomogenizer.[RinsedouncewithH2O,EtOHtoclean.RinsedouncewithEBBbeforeusing.]Homogenizecellswith5pestlestrokes(upanddownisonestroke).Avoidintroducingair.TransfertocleanTOMYtube.
  10. Add150uL5MNaCl(finalconcentration~0.3M).Incubateatroomtemperaturefor5min.
  11. Add5mL1xEBB-plus(containing5%glycerol,0.1mMDTTand150ug/mLBSA).Thus,finalconcentrationsin7.5mLlysateare0.1MNaCl.Incubateatroomtemperaturefor45min.
  12. Meanwhile,thawmicrotubule"seeds"andputat37oCfor30min.Addtaxolto10uM(e.g.1ul0.13mMtaxolto15ulMTseeds)andincubateanadditional15min.
  13. Remove250ulsampleforTOTALMATERIAL("1T").Keepthistubeatroomtemperature,soastobecomparabletotheothersamples.DivideremaininglysatebetweenEppendorfstubes.
  14. CLARIFYLYSATES
  15. Spineppendorftubesat15Kfor20min.Poolsupernatantsina15mLconicaltubeandmixgently.Aliquotlysatetomicrofugetubes(between800uland1ml).Spinmicrofugetubesat15Kfor20min.
  16. Remove250ulTOTALSUPERNATANT("1TS")toafreshtube.
  17. Remove500ulclarifiedlysatefromeachmicrofugetubetoafreshtube.Add5ul1mMtaxol(inDMSO)toeachtube-finalconcentrationis10uMtaxol.Mixgently.
  18. Forastandardtitrationofbindingactivity,addmicrotubulestoeachtubeindecreasingamount.Normally,8,4,2,1,0.5and0uls(MTsmadefrom~3-6mg/mlPCbovinetubulin).For0.5ul,makea10-folddilutionofMTsinBRB80/30%glycerolbuffer(containing10uMtaxol).Allowbindingtoproceedatroomtemperaturefor15min.
  19. Pelletmicrotubule/minichromosomemixat15Kfor8min.ItshouldbepossIBLetoseethelargerMTpellets.
  20. Remove250ulofeachsupernatanttofreshtube.ThesearetheSUPERNATANTFRACTIONS.AspirateallresidualliquidwithdrawnoutPasteurpipets.
  21. ResuspendMTpelletsin250ulof1xEBB(containing0.1mMDTT,100mMNaCl,5%glyceroland1mg/mlBSA,butNOPMSF!).ThesearethePELLETFRACTIONS.NOTE:Ultimately,PELLETswillbe2xconcentratedrelativetoSUPs.
  22. Add250ulof2xASSAULTbuffer(containingtRNAandøX174)toallsamples.Removeproteinbyadding20ulsofProteinaseKsolution(15mg/mlfromBoehringerMannheim)toeachtube.Incubatetubesat50oCfor~1-1.5h(longerisbetter).
  23. AfterProteinaseKtreament,precipitateDNAbyadding250ul6MNH4OAcand700mlisopropanol.Storeat-20oCovernight.
  24. SpindownDNAat15K,30min.4oC.Carefullyaspirateordecantsupernatants,removingasmuchsupernatantaspossible.NOTE:The"PELLETSAMPLE"precipitatedpelletsareoftensmall.WashallDNApelletswith100-200ul70%ethanol.Spinbriefly(~5min)andaspiratetheethanolsupernatant.AllowDNApelletstoairdry.Donotinverttubes.
  25. ResuspendallDNApelletsin30ulTE,incubatingatroomtemperatureor4oCforseveralhours.
  26. PREPARATIONOFSAMPLESFORSOUTHERNANALYSIS.
  27. Remove15ulofeachsampletoafreshtube.Add1ulDNase-freeRNase(BoehringerMannheim,1:1000dilutionof2U/ulstock)to1T,1Ts,andallSUPERNATANTfraction.ThePELLETfractionsdonothavetobe"RNased".Incubateatroomtemperatureforatleast30min.Addsamplebuffertoalltubes.
  28. Loadsamplesontoa0.6%agarosegelcontainingEtBrat0.2ug/ml.Includeasmallamountofsupercoiledtestplasmid(e.g.pDK370)intheDNAsizestandardstoserveasapositivecontrolforhybridization.Thefinalconcentrationofsupercoiledplasmidshouldbesuchthat~0.1ngisloaded.Rungelat20or30Vovernight.
  29. Photographgel.NotewhethertheintensitiesoftheøX174bandsareeveninalllanesandwhether2ucircleDNAisvisibleintheSUPERNATANTfractions.
  30. ProcessgelfortransfertoGeneScreenPlusaccordingtothePosiblotprotocol.Transferforatleast90min.UVcrosslinkDNAtomembrane.Airdry.
  31. HYBRIDIZATION.
  32. Prehybridizeblotat65oCfor~3hinChurchbuffercontaining0.5mg/mldenaturesalmonspermDNA(usually14mlChurchbufferplus0.7ml10mg/mlSSDNAperblot).Addprobe105cpmsperlane)andhybridizeat65oCovernight(atleast18h).Washblot2xwithBuffer1,15mineachat65oCand2xwithBuffer2,15mineachat65oC.Monitortheblot--thelastwashmaynotbenecessary.NOTE:Usethe0.9kbSmaI/PstIURA3fragmentfrompDK377tovisualizepDK370orotherURA3-containingplasmids.Usethe1.8kbSalI/ClaILEU2fragmentfrompDK255tovisualizeYCp41orotherLEU2-containingplasmids.Usethe~2kbXhoI/SalIfragmentfromCV13tovisualizeendogenous2ucircleDNA.Note:makeuptheYWB,EBB2Xassaultbufferfresheachexperiment.
    YWBper10ml5mL2MSorbitol(ifNZarrested,add40uL1.5mg/mL0.336mL1MK2HPO4N2to4mLYWB)0.064mL1MKH2PO44.6mLdH2OYWB,glycerol,PMSF5mL2MSorbitol0.336mL1MK2HPO40.064mL1MKH2PO40.5mL100%ultrapureglycerol0.05mL0.2MPMSF4.05mLdH2O
    1XEBB,glycerol,PMSF(5mL)1mL5XEBB0.25mL100%glycerol12.5uL0.2MPMSF3.75uLdH2O1XEBB,glycerol,DTT,BSA(10mL)2mL5XEBB0.5mLglycerol10uL100mg/mLBSA10uL0.1MDTT7.48mLdH2O
    1XEBB,DTT,NaCl,BSA5mL1mL5XEbb5uL0.1MDTT0.1mL5MNaCl50uL100mg/mLBSA0.25mLglycerol3.6mLdH2O2mLEBB0.2mLNaCl0.1BSA
    REAGENTS
    YWB:50mL2Msorbitol16.8mL0.2MK2HPO43.2mL0.2MKH2PO430mLH2O0.1MDTT
    0.2MPMSFinethanolorispropanolGlusulase
    5XEBB:5mL1MMgCl25mLTRIS-HCl,pH7.40.1mL0.5MEDTA90mLH2O5MNaCl
    1mMtaxolinDMSO2XASSAULTBUFFER:5mL10%SDS,UltraPure5mL0.5MEDTA5mLHEPES-KOH,pH7.6[7.5w/NaOH]85mLH2O**2XA.B.canbefrozenin10mLaliquots.[3.5mLAssaultbuffer,0.5mLtRNA/øX174]
    tRNA/øX174DNA:1mL1mg/mLtRNAinH2O0.1mL10ug/mLuncutøX174DNA5XBRB80100mLs5Xstock80mMPipespH6.8(KOH)10mL800mMPipespH6.8(KOH)1mMEGTA1ml100mMEGTA1mMMgCl20.1mL1MMgCl2
    MINICHROMOSOME-MICROTUBULEBINDINGASSAY
  33. GROWTHOFCELLS.Grow100mLofcellstoOD600=0.7-0.8at23oC.Forgoodbindingactivityithasprovedimportanttomaintaincellsinlogphase.Normally,2dayspriortodayofexperiment,asinglemedium-sizedcologyispickedfromselectivemediumandinoculatedinto5mLYPS(or-URA,asIdo).Twoadditionaldilutionsaremadefromthis"neat"inoculum(e.g.1:10and1:25).Thegoalistohavelatelogphaseculturesthenextday(~4x107cells/mL).
  34. Thedaypriortoexperiment,makea1:10dilutionofthesuitable5mLovernight.DeterminetheOD600andcorrelatingcelldensityfromthechart.Setupthree100mLYPDculturesatthefollowingdensities:~0.7x105,1x105and1.5x105cells/mL.
  35. Harvestcellsintwo50mLconicaltubes,1.5-2.0Kfor8-10min.WashwithH2Oandconsolidatecellsinonetube.Re-spin.NOTE:Ifcellshavebeenarrestedwithnocodazoleorafactor,includetheinhibitorintheH2Owash.
  36. SPHEROPLASTING.
  37. Re-suspendcellsin4mLYWB(containing10mMbeta-mercaptoethanoland1mMPMSF).Lesitatroomtemperaturefor2-3min.Add100uLglusulase.Incubateat23oCwithgentleshakingtoformspheroplasts.Spheroplstingshouldbecompletewithinonehour;avoidincubationslongerthan1h,15min.NOTE:Ifcellshavebeenarrestedwithnocodaoleoralphafactor,includetheinhibitorintheYWB.
  38. TransferspheroplastedcellstoaTOMYtube.Spinat3.5Kfor7min.Re-suspendcellsin4mLYWB(containing1mMPMSFonly)bygentlypipettingupanddown,usinga1mLplasticpipet.Re-spin.Repeatwashprocess,foratotaloftwowashes.NOTE:Ifcellshavebeenarrestedwithnocodazoleoralphafactor,includetheinhibitorintheFIRSTWASHONLY.
  39. Gentlyreuspendcellsin2.5mL1XEBB(containing0.1mMDTTand0.5mMPMSF).Transfertohomogenizer.
  40. Allowcellsto"swell"for10minatroomtemperature,thenhomogenizecellswith5pestlestrokes(upanddownisonestroke).Avoidintroducingair.TransfertocleanTOMYtube.
  41. Add150uL5MNaCl(finalconcentration~0.3M).Incubateatroomtemperaturefor5min.
  42. Add5mL1XEBB-plus(containing0.1mMDTT,150ug/mLBSA,and0.5mMPMSF).Thus,finalconcentrationsin7.5mLlysateare0.1MNaCland100ug/mLBSA.Incubateatroomtemperaturefor45min.
  43. Thawmicrotubule"seeds"andputat37oCfor30min.Addtaxolto10uM(e.g.1uL0.13mMtaxolto12uLMTs)andincubateanadditional15min.
  44. Remove250uLsampleforTOTALMATERIAL(so-called"1T").Keepthistube(and1Ts,seebelow)atroomtemperature,soastobecomparabletotheothersamples.DivideremaininglysatebetweentwoTOMYtubes.
  45. CLARIFYLYSATES.
  46. SpinTOMYtubesat15Kfor20min.Poolsupernatantsina10mLconicaltubeandmixgently.Aliquotlysatetomicrofugetubes.Theminimumnumberoftubesis[1thenumberofbindingreactionsplanned](usually0.7-1mLextractpertube).Spinmicrofugetubesat15Kfor20min.
  47. Remove250uLSTARTINGMATERIAL("1TS")toafreshtube.
  48. Remove500uLclarifiedlysatefromeachmicrofugetubetoafreshtube.Add5uL1mMtaxol(inDMSO)toeachtube-finalconcentrationis10uMtaxol.Mixgently.
  49. Forastandardtitrationofbindingactivity,addmicrotubulestoeachtubeinincreasingamount.Normally,0,0.2,0.5,1,2,and4uLFor0.2uLand0.5uL,makea10-folddilutionofMTsinBRB80/30%glycerolbuffer(containing10uMtaxol).Allowbindingtoproceedatroomtemperaturefor15min.
  50. PelletMTs(andassociatedminichromosomes)at15Kfor8min.ItshouldbepossibletoseethelargerMTpellets(usuallybluish-white).
  51. Remove250uLofeachsupernatanttofreshtube.ThesearetheSUPERNATANTFRACTIONS.AspirateallresidualliquidwithdrawnoutPasteurpipets.
  52. ResuspendMTpelletsin250uLof1XEBB(containing0.1mMDTT,100mMNaCl,and1mg/mlBSA,butNOPMSF!).ThesearethePELLETFRACTIONS.NOTE:Ultimately,PELLETswillbe2XconcentratedrelativetoSUPs.
  53. Add250uLof2XASSAULTbuffer(containingtRNAandøX174DNA)toallsamples(i.e.allsupsandpellets,aswellas1Tand1Ts).Removeproteinbyadding5mLofProteinaseKsolution(20mg/mLfromBoehringerMannheim)toeachtube.Incubatetubesat50oCfor~1-1.5h.(longerisbetter).
  54. AfterProteinaseKtreatment,precipitateDNAbyadding250uL6MNH4OAcand500uLisopropanol.Storeat-20oCovernight.
  55. SpindownDNAinTOMYat15K,30min.4oC.Carefullyaspirateordecantsupernatants,removingasmuchsupernatantaspossible.NOTE:The"PELLET"pelletsareoftensmall.WashallDNApelletswith100-200uL70%ethanol.Spinbriefly(~5min.)andaspiratetheethanolsupernatant.AllowDNApelletstoairdry.Donotinverttubes.
  56. ResuspendallDNApelletsin30uLTE,incubatingatroomtemperatureor4oCforseveralhours.
  57. PREPARATIONOFSAMPLESFORSOUTHERNANALYSIS.
  58. Remove15uLofeachsampletoafreshtube.Add1uLDNase-freeRnase(BoehringerMannheim,1:1000dilutionof2U/uLstock)to1T,1Ts,andallSUPERNATANTfractions.ThePELLETfractionsdonothavetobe"RNased".Incubateatroomtemperatureforatleast30min.Addsamplebuffertoalltubes.
  59. Loadsamplesontoa0.6%agarosegelcontainingEtBrat0.2ug/mL.Includeasmallamountofsupercoiledtestplasmid(e.g.pDK370)intheDNAsizestandardstoserveasapositivecontrolforhybridization.Thefinalconcentrationofsupercoiledplasmidshouldbesuchthat~0.1ngisloaded.Rungelat20or30Vovernight.
  60. Photographgel.NotewhethertheintensititesoftheøX174bandsareeveninalllanesandwhether2ucircleDNAisvisibleintheSUPERNATANT(hopefully)fractions.
  61. ProcessgelfortransfertoGeneScreenPlusaccordingtothePosiblotprotocol.Transferforatleast90min.UVcrosslinkDNAtomembrane.Airdry.
  62. HYBRIDIZATION.
  63. Prehybridizeblotat65oCfor~3h.inChurchbuffercontaining0.5mg/mLdenaturedsalmonspermDNA(usually14mLChurchbufferplus0.7mL10mg/mLSSDNAperblot).Addprobe(105cpmsperlane)andhybridizaeat65oCovernight(atleast18h.).Washblot2XwithBuffer1,15min.eachat65oCand2XwithBuffer2,15min.eachat65oC.Monitortheblot-thelastwashmaynotbenecessary.NOTE:Usethe0.9kbSma1/Pst1URA3fragmentfrompDK377tovisualizepDK370orotherURA3-containingplasmids.Usethe1.8kbSal1/Cla1LEU2fragmentfrompDK255tovisualizeYCp41orotherLEU2-containingplasminds.Usethe~2kbXho1/Sal1fragmentfromCV13tovisualizeendogenous2ucircleDNA.
    YWBforspheroplasting:5mLYWB25uL0.2MPMSF3.5uLbeta-MEYWBforwash:8mLYWB40uL0.2MPMSF
    1XEBB(tobreak):2mL5XEBB25uL0.2MPMSF10uL0.1MDTT8mLH2O1XEBB-plus(todilute):1mL5XEBB12.5uL0.2MPMSF5uL0.1MDTT75uL100mg/mlBSA4mLH2O
    1XEBB-doubleplus(forMTpellets):1mL5XEBB12.5uL0.2MPMSF5uL0.1MDTT50uL100mg/mlBSA100uL5MNaCl3.9mLH2O2XAssaultBuffer:XmL2XASSAULTBUFFER(X=0.25mLx#samples)0.1xXmLtRNA/øX174DNA
    3.75dH2O0.25EDTA
    0.25SDS0.25HEPES
    0.5tRNAøX174

免责声明 本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘”。违反上述声明者，本网将追究其相关法律责任。
发布于 ： 2018-12-26 阅读（221）
▍ 相关分类
缓冲液
酸碱缓冲液 上样缓冲液 电泳缓冲液 其它缓冲液
生物分子
氨基酸 核苷酸 脂类 固醇类 糖类 蛋白交联剂 储存液 反应底物 小分子 受体 转运体 激素 脂质体
染色剂
荧光染料 其他生物染料
其他
酸 碱 盐 醇 酮 酚 胺 酯 微球 磁珠 琼脂糖 发光剂 去垢剂 多聚物 表面活性剂 其他试剂 单体
标准品
微球标准品 荧光染料标准品 生化标准品
脂质代谢研究
▍ 相关文章
豪沃生物科技（上海）有限公司 KCF系列快开式静密封磁力搅拌反应釜结构及使用简介 jz.docin.com... 细胞因子的3种作用方式 葡萄糖6磷酸脱氢酶荧光斑点试验_葡萄糖6磷酸脱氢酶荧... 考马斯亮蓝R250及G250染料 Relia Tech 代理（5）_一级代理品牌 B淋巴细胞膜表面免疫球蛋白的检查实验_直接免疫荧光法 美国Thermo Scientific™ Applied Biosystems 7500实时荧光 ... 谁收藏这个实验:性染色质检测实验 实验方法 人乙型肝炎表面抗原(HBsAg)ELISA分析试剂盒说明书elisa试剂盒原... Diabetes Care:良好的血糖和血压控制有益于延长T2DM患者生存期... : Tying Ligatures and Sutures(Instrument Tie) 实验方法 丁 ...
▍ 相关问答
目前荧光免疫产品中多数用的是荧光素还是荧光微球还是荧光染料 ...
请教可以长时间染活细胞的荧光染色剂_问答详情_荧光染料_染色剂...
pi染料激发波长_完美作业网_www.wanmeila.com
荧光染料选择和数据分析
荧光显微镜用什么染料较好_这样
怎样检测荧光剂 – 糗问
纺织科学技术:染整技术试题及答案(每日一练) 考试题库 91考试网
活体成像dir染料与cy5.5荧光染料哪个好– 960问答
水性荧光绿染料 切削液添加剂 洗手液洗涤剂环保色粉水溶性荧光剂
在流式细胞术分析的荧光染料中,何为背景荧光
请问PCR常用荧光染料的化学结构是什么?
【求助】共聚焦荧光染料选择问题_问答详情_荧光染料_染色剂_...
自营商城图标
自营商城
一手货源 13%专票
正品保障图标
正品保障
全球直采 在线追溯
解放采购图标
解放采购
在线下单 简单省事
及时交付图标
及时交付
限时必达 畅选无忧
版权所有 : ©2005- 苏州蚂蚁淘生物科技有限公司
公司邮箱 : info@ebiomall.com
联系电话 : 4000-520-616

© 2005- 苏州蚂蚁淘生物科技有限公司 版权所有。 苏ICP备17049038号-11 苏公网安备 32059002001627号

声明：本网站部分产品受外界条件影响（包含但不限于进口国出口国政策限制等因素），无法提供销售服务，下单后，会由客服人员联系厂家确认后另行通知。部分危险品无法清关，请勿下单

友情链接
首页
分类
品牌中心
购物车
我的