inoue法制备超级感受态细胞-丁香通

《inoue法制备超级感受态细胞-丁香通》由会员上传分享，免费在线阅读，更多相关内容在行业资料-天天文库。

1、Inoue法制备大肠杆菌超级感受态细胞关键词： Inoue法 大肠杆菌 感受态2008-07-2100:00 来源：互联网 点击次数：1704实验步骤：1、InoculatefromanovernightgrowninLB.从培养过夜的LB平板上挑取单菌落。2、Growin250ml"SOB"at18℃untilOD600 =0.6.(0.3)接种于250mlSOB，18度培养至OD＝0.6。3、Onicefor10minutes.菌液置冰上10分钟。4、Spinat2500xg(5000rpminaSorvallGSAor3000rpminaBeckmanJ-

2、6Bcentrifuge)for10min.at4℃.4度2500g离心10分钟。5、Resuspendcellsgentlyin80mloficecold"TB".小心用80ml预冷TB重悬细胞。6、Onicefor10minutes.(30min)菌液置冰上10分钟。7、Spinat2500xg(5000rpminaSorvallGSA,5500rpminaSorvallSS-34,or3000rpminaBeckmanJ-6Bcentrifuge)for10min.at4℃.4度2500g离心10分钟。8、Resuspendcellsgentlyin20m

3、loficecold"TB".小心用20ml预冷TB重悬细胞。9、AddDMSOtoafinalconcentrationof7%.加入DMSO至终浓度7%。10、Placeonicefor10minutes.置冰上10分钟。11、Aliquotinto1-2mlandfreezeinliquidnitrogen.分装，液氮速冻。12、Storeinliquidnitrogen.冻存。SOBMediumandTB(TransformationBuffer)SOB 2%(w/v)bactotryptone TB 0.5%(w/v)yeastextract 10mM

4、 Pipes 10mM NaCl 55mM MnCl2 2.5mM KCl 15mM CaCl2 10mM MgCl2 250mM KCl 10mM MgSO4 在加入MnCl2之前先用5NKOH调pH值到6.7，adjustpHto6.7with5NKOHpriortoaddingtheMnCl2 thankstoMarkusSchneemannforthetip! pH6.7-7.0 Note:Competentcellsarefragile(cellwallisthoughttobeweakened),thereforetreatthecellsgentl

5、ywhenpreparingthesecells.Donotvortexorpippetteupanddowntoresuspendthecells.Donotspinthecellsattoogreataspeed(spinningdownat5000gwillcausesomecellstolyse).Alwayskeepthecellschilledwhenmakingcompetentcell.Donotletthemwarmup.Freezingthecellsappeartomakecellsmorecompetent.Somecellstrains

6、mayworkbetterthanothers(DH5alphaworkswellinmyhand).Notealsothatsomecells(e.g.HB101)hasgreaterrecombinationactivitythanothers.Thismethoddoesn'tappeartoworkwithBL21,sojustgrowthecellsat30or37℃whenmakingBL21competentcells.However,ithasbeensuggestedthattheefficiencyofBL21preparedusingIno

7、uemethodmaybeimprovedbytreatingitwithDTTbeforefreezing(addto3.5%v/vofa2.2MDTT,10mMKAcpH6solutionandincubate10minutesonice).Heatshocktimeshouldbedeterminedfordifferentstrainsofcell.ForDH5alphaorJM109use30-45sec.ForBL21use120sec.Deactivateligasepriortotransformation.Ligasemayreducetran

8、sformationef