1、感受态细胞制作方法七:Inoue法制备超级感受态细胞越干净越好,越冷越好。1、将洗干净的瓶子(500ml三角烧瓶)用Milli-Q水浸泡2-3小时,倒去烧瓶中的水并将烧瓶倒扣在吸水纸上2-3分钟。用Milli-Q水配置250ml LB液体培养基。高压灭菌。2、准备两盒1.5ml EP管,每盒中放置两张吸水纸,共约两百个。高压灭菌。3、于早晨7-8点钟小摇菌种,挑取单菌落于5ml Milli-Q水配置且灭菌的LB液体培养基(无抗性)中,37培养12小时以上(OD600大于1.5)。可以用一个新的50ml 进口离心管(costa,BD等品牌都可以)来摇细菌。4、晚上10点钟左右,按1:100大摇细
2、菌。超静台内吸取2.5ml小摇细菌于高压灭菌的250ml SOB培养基(无抗性)中, 18-22,200rpm摇过夜。5、第二天上午测量细菌OD600值。Top10, JM109等生长速度快的细菌约在上午9点左右OD600达到0.55左右,DH5则要到下午4-6点钟(可以多摇几瓶细菌,梯度接菌,如1:50,1:100,1:200等)。6、将OD600达到0.55的细菌置于冰上10min(OD600在0.4-0.8之间都可以,对结果影响不大)。7、4,4000rpm(2500g),10min集菌(用新的50ml 进口离心管)。8、超静台内弃上清,将管倒扣在事先灭好的吸水纸上,大力向下击打,尽可能
3、去掉剩余SOB(LB)。9、每管倒入10ml 预冷的Inoue转化缓冲液,拧紧盖子。在冰上来回滑动重悬细菌(重悬的时间与向下击打的力度和来回滑动的速度相关,这两者与感受态效率没有直接关系)。10、超静台内向每管倒入约30ml 预冷的Inoue转化缓冲液,来回颠倒混匀,冰上10min。11、4,4000rpm,10min集菌。12、超静台内弃上清,将管倒扣在事先灭好的吸水纸上(换一张吧,别用刚才那张),大力向下击打,尽可能去掉剩余缓冲液。13、每管倒入10ml 预冷的Inoue转化缓冲液,拧紧盖子。在冰上来回滑动重悬细菌。 14、轻轻来回混匀(记住要轻轻)。置于冰上10min。 15、将冰浴的感
4、受态细胞分装到事先灭菌后并放到-20或4的EP管中,一个一个丢到液氮罐里,然后冻到-80(建议100l每管,传说液氮速冻可以提高5倍的感受态效率)。 感受态效率在108-107不等。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2008-07-21 00:00来源:互联网点击次数:1515实验步骤:1、Inoculate from an overnight grown in LB.从培养过夜的LB平板上挑取单菌落 。2、Grow in 250 ml SOB at 18 until OD600= 0.6.(0.3)接种于250ml SOB,18度培养至OD0.6。3、O
5、n ice for 10 minutes.菌液置冰上10分钟。4、Spin at 2500 x g (5000 rpm in a Sorvall GSA or 3000 rpm in a Beckman J-6B centrifuge)for 10 min.at 4.4度2500g离心10分钟。5、Resuspend cells gently in 80 ml of ice cold TB.小心用80ml预冷TB重悬细胞。6、On ice for 10 minutes.(30min)菌液置冰上10分钟。7、Spin at 2500 x g (5000 rpm in a Sorvall GSA,
6、5500 rpm in a Sorvall SS-34,or 3000 rpm in a Beckman J-6B centrifuge)for 10 min.at 4.4度2500g离心10分钟。8、Resuspend cells gently in 20 ml of ice cold TB.小心用20ml预冷TB重悬细胞。9、Add DMSO to a final concentration of 7%.加入DMSO至终浓度7%。10、Place on ice for 10 minutes.置冰上10分钟。11、Aliquot into 1-2 ml and freeze in liqui
7、d nitrogen.分装,液氮速冻 。12、Store in liquid nitrogen or -80oC.冻存。SOB Medium and TB (Transformation Buffer)SOB2% (w/v) bacto tryptoneTB 0.5% (w/v) yeast extract10 mMPipes10 mMNaCl55 mMMnCl22.5 mMKCl15 mMCaCl210 mMMgCl2250 mMKCl10 mMMgSO4在加入MnCl2之前先用5N KOH调pH值到6.7,adjust pH to 6.7 with 5N KOH prior to addi
8、ng the MnCl2thanks to Markus Schneemann for the tippH 6.7 - 7.0Note:Competent cells are fragile (cell wall is thought to be weakened),therefore treat the cells gently when preparing these cells.Do not vortex or pippette up and down to resuspend the cells.Do not spin the cells at too great a speed (s
9、pinning down at 5000g will cause some cells to lyse).Always keep the cells chilled when making competent cell.Do not let them warm up.Freezing the cells appear to make cells more competent.Some cell strains may work better than others (DH5alpha works well in my hand).Note also that some cells (e.g.H
10、B101)has greater recombination activity than others.This method doesnt appear to work with BL21,so just grow the cells at 30 or 37 when making BL21 competent cells.However,it has been suggested that the efficiency of BL21 prepared using Inoue method may be improved by treating it with DTT before fre
11、ezing (add to 3.5% v/v of a 2.2M DTT,10mM KAc pH6 solution and incubate 10 minutes on ice).Heat shock time should be determined for different strains of cell.For DH5alpha or JM109 use 30-45 sec.For BL21 use 120 sec.Deactivate ligase prior to transformation.Ligase may reduce transformation efficiency
12、.Diluting the ligation mixture (5x)can also increase transformation efficiecy by reducing the amount of reagents/contaminants that may affect transformation.Likewise it has been suggested that phenol/chloroform treatment may also increase efficiency,but it is probably too much trouble to bother tryi
13、ng.The DNA added should not be more then 5% of the volume of competent cells used.The final DNA concentration should not exceed 5 ng/l.The method above should give a transformation efficiency of more than 108 cfu per g of plasmid DNA (pUC or pBluescripts)with over 109 cfu possible.Transformation eff
14、iciency has a roughly inverse relationship with the size of plasmids.Cells with deoR mutaion (e.g.DH5alpha)can improved the transformation of large plasmid.Relaxed plasmids has 3/4 of the transformation efficiency of supercoiled plasmid.2 different plasmids can be transformed at the same time,or one
15、 after another.But they must be compatible (they cannot have the same replicon).For routine transformation whereby efficiency of transformation is of no import,some of the steps may be shorten or omitted.For example,heat-shock step may be unnecessary and recovery incubation time at 37 can be reduced
16、 or omitted (but do note that this may depends on the antibiotic used for selection-for ampicillin-type antibiotics the incubation time is not really that important,therefore you can plate the cells straight after heat-shock if you wish.for other antibiotics,however,the incubation time may be essent
17、ial).Plating cells-dry 1.5% agar plates (exposed upside down)at 37 for 2-4 hours just before use,the plate should be able to soak up to 0.8-1 ml of media when plating.For blue-white selection,it is not necessary to make X-gal plate,just add X-gal+IPTG direct to cells,mix and then plate.Detergents ma
18、y be detrimental to the transformability of the competent cells,therefore the glassware used for making competent cells should not be washed with detergents.Polycarbonate flask may also be used instead of glass flask.DMSO can dissolve polystyrene,therefore use polypropylene tubes.When cloning diffic
19、ult and less stable sequence (e.g palindrome,repeats,LTR sequences),it helps to grow transform cells at lower temperatures (25-30 or room temperature)in very rich media (e.g.Terrific Broth).Also terminate growth before reaching late stationary growth phase when grown in liquid media (i.e harvest cel
20、ls at OD550between 1 and 2).Use of stabilizing strain is also useful.There are other methods of making competent cells-e.g.CaCl2method,RbCl method which is more effective than CaCl2method.Electroporation is supposed to give higher efficiency (up to 1010 transformants per g plasmid claimed),but for t
21、he simple cloning that we do,its use is not warranted (and its more expensive,more trouble than its worth,etc.).If a cooling shaker is not available-grow the cells at room temperature.More discussions on making competent cell as well as references can be found in TIBS articles Preparing ltra-compete
22、nt E.coli and Better competent cellsIt is also possible to transform cells straight from plate.It is convenient but you should expect low efficiency.See the following reference for more details (as well as more information on competent cells and other protocols):本文引自:Hanahan et al,Methods in Enzymol
23、ogy 204,63大肠杆菌感受态细胞的几种制备方法感受态细胞(Competent cells) :常态的细胞不能摄入外部溶液中的DNA,所以要转化质粒DNA进入大肠杆菌必须首先制备感受态的大肠杆菌细胞。受体细胞经过一些特殊方法(如:CaCl,RuCl等化学试剂法)的处理后,细胞膜的通透性发生变化,成为能容许多有外源DNA的载体分子通过的感受态细胞(competent cell) 。 转化:是将异源DNA分子引入一细胞株系,使受体细胞获得新的遗传性状的一种手段,是基因工程等研究领域的基本实验技术。 进入细胞的DNA分子通过复制表达,才能实现遗传信息的转移,使受体细胞出现新的遗传性状。转化过程所
24、用的受体细胞一般是限制修饰系统缺陷的变异株,即不含限制性内切酶和甲基化酶的突变株。 -互补现象:因为许多载体都带有一个LacZ基因的调控序列和头146个氨基酸的编码信息,编码-互补肽,该肽段能与宿主编码的缺陷型-半乳糖苷酶实现基因内互补(-互补)。当这种载体转入可编码?半乳糖苷酶C端部分序列的宿主细胞中时,在异丙基-D硫代半乳糖苷(IPTG)的诱导下,宿主可同时合成这两种肽段,虽然它们各自都没有酶活性,但它们可以融为一体形成具有酶活性的蛋白质。所以称这种现象为-互补现象。由互补产生的-半乳糖苷酶(LacZ)能够作用于生色底物5-溴-4-氯-3-吲哚-D-半乳糖苷(X-gal)而产生蓝色的菌落,
25、所以利用这个特点,在载体的该基因编码序列之间人工放入一个多克隆位点,当插入一个外源DNA片段时,会造成LacZ()基因的失活,破坏-互补作用,就不能产生具有活性的酶。所以,有重组质粒的菌落为白色,而没有重组质粒的菌落为蓝色。方法一:TSS方法制备感受态细菌(又称一步法)一、准备工作1、缓冲液1TSS的配制事先配制1M的氯化镁:20.3g氯化镁(6分子水结晶),定容于100ml去离子水后封装,不用灭菌。取干净的100ml 量筒和100ml 烧杯,用量筒量取100ml去离子水,加入至烧杯中,取 1 g 蛋白胨,0.5g 酵母抽提物,0.5g 氯化钠,10 g PEG(MW= 3350),5ml D
26、MSO,5ml 的1 M氯化镁,溶解后用盐酸或者氢氧化钠调整pH为6.5,混匀后用0.22um 滤器过滤除菌。储存在4度,保质期约6个月。2、已划平板(或者用枪头沾取并稀释后涂布)并在培养箱中过夜培养的菌种Dh5,用于接种并振荡培养。两个锥形瓶,分别装有30 ml和50ml LB,灭菌后置于4冰箱备用。3、若干已灭菌的琼脂平板,一个未加抗生素,用于第二步的接种,其余做转化用。 二、感受态制备程序前一天晚上调单菌落至30 ml LB中过夜培养(12-16h),按1:100 比例将过夜培养的菌液(500ul)加入到新鲜的50 ml LB培养液中,于37 振荡培养至OD600约为0.4(培养时间2h
27、4050min)。冰浴30分钟后4度1000g离心10min,弃上清,收获细菌。加入原体积十分之一(这里为5 ml)的1 TSS 液(冰预冷)悬浮细胞,然后分装成100l/份,全部冰上操作,-80保存。三、转化取一管(100l)感受态细菌,(冻存细胞应置于冰上缓慢融化后立即使用)加入3-5l DNA(0.1100ng),轻轻混匀后冰浴30min。加入0.9ml含20mmol/L葡萄糖的LB培养液,37度150r/min温和振荡培养 60min,涂布,室温放置约20分钟后放于37度恒温培养箱过夜培养(17-20h)。方法二:CaCl2法细菌转化的方法多以Mendel和Higa(1970)的发现为
28、基础,其基本方法是用冰预冷的CaCl2或多种2价阳离子等处理细菌,使之进入感受态得以转化。 用CaCl2制备新鲜或冷冻的大肠杆菌感受态细胞,常用于成批制备感受态细菌。本法适用于大多数大肠杆菌菌株,且迅速、重复性好。1从37培养1620h的新鲜平板中挑取一个单菌落(如大肠杆菌DH5),或1ml新鲜的1620h过夜培养物,转到一个含有100mlLB培养基的1L或500ml烧瓶中。于37剧烈振摇培养约23h(旋转摇床200300r/min),每隔2030min测量OD600值0.4。 2在无菌条件下将细菌转移到一个,用冰预冷的50ml聚丙烯离心管中,在冰上放置1020min。 3于4用Sorvall
29、GS2转头(或与其离心管相配的转头)以4000r/min离心10min,以回收细胞。 4倒数培养液,将管倒置1min以使最后残留的痕量培养液流尽。5以10ml用冰预冷的0.1mMCaCl2重悬每份沉淀,放于冰上10min。6于4用SorvallGS3转头(或与其相应的转头)以4000r/min离心10min,以回收细胞。 7倒出培养液,将管倒置1min以使最后残留的痕量培养液流尽。8每50ml初始培养物用2ml冰预冷的0.1M CaCl2重悬每份沉定,此时,可以迅速将细胞分装成小份,液氮中冰冻,-70贮存备用。9用冷却的无菌吸头从每种感受态细胞悬液中各取200l转移到无菌的微量离心管中,每管加
30、DNA或连接反应混合物(体积10l,DNA50ng),轻轻旋转以混匀内容物,在冰中放置30min。10将离心管放到预加温到40的循环水浴中的试管架上,放置90s2min,不要摇动试管。11快速将管转移到冰浴中,使细胞冷却12min。12每离心管加800l SOC培养基,用水浴将培养基加温到37,然后将管转移到37摇床上,温育45min使细菌复苏,并且表达质粒编码的抗生素抗性标记基因。13将适当体积(每个90mm平板可达200l)已转化的感受态细胞转移到含200mmol/l MgSO4和相应抗生素的SOB培养基上。14将平板置于室温至液体被吸收。15倒置平皿,于37培养,1216h后可出现菌落。
31、方法三: CAS super One-Step Competent Cell Preps Kit采用一种简单便捷的方法处理大肠杆菌,使其成为感受态细胞,便于质粒DNA的转化,可满足一般实验的要求。与CaCl2法相比具有多种优点:使用方便,只需一步即可完成感受态细胞的制备;转化效率略高于CaCl2法,一般可达106-108cfu/g,满足一般实验需要;感受态细胞制成后即可保存于-70,无须加入甘油,并且经长期保存活力无明显下降。 一、准备工作1 从液氮或低温冰箱中取出的大肠杆菌冻存菌,在LB平板上划线,37过夜至长出单菌落。挑形态饱满的单菌落2个,分别接种5ml LB培养基中,37,250rpm,过夜培养。2 次日从5ml LB培养物吸取200l转入50ml LB培养基中(250ml 锥形瓶),37,250rpm培养2小时,此时OD600约0.40.5。二、感受态细胞的制备3 吸取1ml菌液到1.5ml离心管里,5000rpm离心5分钟,弃去上清。 4 加入100l预冷的Solution A,悬浮菌体,冰浴30分钟。 5 此时感受态细胞已经制成可立即使用或-70保存。 三、细胞转化6 在感受态细胞中加入100pg-1
copyright@ 2008-2022 冰豆网网站版权所有
经营许可证编号:鄂ICP备2022015515号-1