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分子克隆——蓝白斑筛选 - 知乎

分子克隆——蓝白斑筛选 - 知乎首发于分子克隆切换模式写文章登录/注册分子克隆——蓝白斑筛选BT 时代之前已经介绍过抗生素筛选，可以选择包含目的质粒的菌落，但是如何直接选择成功克隆（目的基因整合入质粒）的菌落呢？蓝白斑筛选是达到此目的的一种广泛使用的方法。01蓝白斑筛选的基本原理研究较为充分的细菌乳糖操纵子结构包含一个编码 -半乳糖苷酶基因lacZ。lac操纵子结构的表达可受乳糖及其类似物如IPTG的诱导。准确的说，IPTG能结合并失活lac操纵子的阻遏蛋白，从而起始lac操纵子的表达。lac操纵子表达后， -半乳糖苷酶能将显色底物X-gal降解为半乳糖和一种不溶的蓝色色素，那么这是如何用于筛选呢？乳糖操纵子结构诱导物：乳糖，其常用类似物为IPTG。LacI：为反式作用因子，编码阻遏蛋白。启动子（Promotor，P）操纵子（Operator，O）：为顺式作用元件，无诱导物时，与反式作用因子即阻遏蛋白结合从而抑制乳糖操纵子的转录。LacZ： -半乳糖苷酶，可切断乳糖的半乳糖苷键生成一分子半乳糖和一分子葡萄糖。LacY： -半乳糖苷乙酰转移酶，将乙酰辅酶A上的乙酰基转移到 -半乳糖苷上。LacA： -半乳糖苷透性酶，将半乳糖苷转运进入细胞中。当环境中有足够的乳糖时，乳糖受本底水平表达的 -半乳糖苷酶作用形成别构糖（直接诱导物，有些地方说是半乳糖，有些地方说是半乳糖苷），与阻遏蛋白结合而改变其构象，使其失去与操纵子结合的能力，基因簇得以转录，合成大量能代谢、利用乳糖的酶。02实验室蓝白斑筛选科学家发现在lacZ上删除一个片段（lacZ∆M15突变）可产生一种无功能的 -半乳糖苷酶。将该序列片段在lacZ∆M15突变株中表达出氨基酸片段，可以补偿基因缺陷，得到有功能的酶，该氨基酸片段为肽，这个过程称为互补。该系统的应用方法如下。将工程化的含多克隆位点（MCS）的肽（下图橙色部分）插入质粒中，构成一个互补克隆质粒。当进行克隆反应时，目的基因（红色）插入到MCS中，打断肽基因（图A）而不能补偿宿主细胞中的 -半乳糖苷酶突变。若未成功克隆则保留了完整的肽，因此宿主细胞中可表达具有功能的 -半乳糖苷酶（图B）。蓝白斑筛选平板培养基如下，含有插入片段的质粒转化入大肠杆菌中只能表达无功能的 -半乳糖苷酶，而使E.coli菌落呈正常的乳白色；相反，含有未整合目的基因质粒的菌由于能表达完整的 -半乳糖苷酶，可将X-gal分解出蓝色色素，从而能长出蓝色菌落。需要注意的是，培养基中除了X-gal也需含有IPTG以确保lac操纵子的转录。03实验过程中的要点1、要有良好的对照。转化空质粒骨架，该平板培养基上的所有克隆应该都是蓝色的，以此证明IPTG和X-gal正常发挥作用。2、足够的培养时间。给平板足够的时间表达完整的 -半乳糖苷酶并将X-gal分解出蓝色色素（16-20h），只有白色菌落的平板是可疑的。3、过夜培养后冰箱内放置平板。过夜培养后在4℃放置几个小时可增加分解出的色素含量，加深阴性克隆的蓝色，以便更好的区分蓝色和白色菌落。4、小心制作平板培养基。X-gal对光和高温敏感，应该在培养基经高压灭菌后再加入X-gal。如果将X-gal涂在先前制备的平板上，要确保其均匀分布，使用平板培养基前要充分干燥。5、识别假阳性克隆。蓝白斑筛选只能确定编码肽的序列是否被干扰失活，不能确定就是目的基因插入MCS。任何干扰肽DNA的错误克隆都会导致形成白色菌落。6、识别假阴性克隆。这种情况较少，但是如果一个小片段插入MCS，表达时可能仍会形成有功能的 -半乳糖苷酶，最终产生蓝色菌落。蓝白斑筛选是一个缩小筛选范围的好方法。7、使用合适的大肠杆菌菌株（含有lacZ ∆M15突变）。如XL1-Blue，DH5 ，DH10B，JM109，STBL4，JM110和Top10等。8、使用合适的质粒（含有互补克隆MCS）。一些常用的载体有pGEM-T，pUC18和pBluescript等。蓝白斑筛选法必须在确保细胞中含有质粒的前提下才能发挥其应有的作用，结合抗生素选择和蓝白斑筛选能确保白色菌落含有成功克隆的质粒，而不是细胞中根本就不含有互补质粒。04其它筛选方法蓝白斑筛选法可能是使用最为广泛的用来筛选含插入片段的目的菌落的方法，除此以外还有一些其他的筛选方法。有些质粒上编码一个致死基因，克隆片段插入到该基因中的MCS则扰乱该致死基因的表达，理论上可以使存活的菌落均含成功克隆的质粒，例如ccdB基因系统。这和蓝白斑筛选中肽的DNA干扰相似。抗生素选择同样被与之结合使用以确保阳性菌落中含有质粒。还有其他的不用抗生素选择来筛选含质粒细胞的方法。这些方法依赖于细胞对培养基特定成分的敏感性或需要，可利用质粒上的基因得以存活。这种质粒可能含有可以利用培养基中一些特定成分的基因，没有这种底物，细胞则不能生长繁殖形成菌落；或者质粒上的某种基因可以改变致死表型，从而使含质粒的菌存活。参考书籍：Plasmids 101:A Desktop Resource —— Created and compiled by Addgene March 2017（3rd Edition）发布于 2020-04-02 19:43赞同 584 条评论分享喜欢收藏申请转载文章被以下专栏收录分

蓝白斑筛选_百度百科

选_百度百科网页新闻贴吧知道网盘图片视频地图文库资讯采购百科百度首页登录注册进入词条全站搜索帮助首页秒懂百科特色百科知识专题加入百科百科团队权威合作下载百科APP个人中心蓝白斑筛选播报讨论上传视频基因工程常用的细菌重组子的筛选方法收藏查看我的收藏0有用+10蓝白斑筛选是一种基因工程常用的细菌重组子的筛选方法。野生型埃希氏大肠菌（E.Coli）产生的β-半乳糖苷酶可以将无色化合物X-gal（5-溴-4-氯-3-吲哚-β-D-半乳糖苷）切割成半乳糖和深蓝色的物质5-溴-4-靛蓝。5-溴-4-靛蓝可使整个菌落产生蓝色变化。在经人工插入外源基因后，突变型大肠杆菌的β半乳糖苷酶基因被插入的外源基因切断，无法形成完整的β半乳糖苷酶，故不能对无色化合物X-gal进行切割，菌落呈白色。中文名蓝白斑筛选外文名Blue-White Screening概述蓝白斑筛选是基因工程操作的一种简介一种基因工程常用的重组菌筛选筛选原理是根据载体的遗传特征筛选重组目录1简介2原理3适用方面4常见问题简介播报编辑蓝白斑筛选(5张)蓝白斑筛选是重组子筛选的一种方法，是根据载体的遗传特征筛选重组子，主要为α-互补 [1]与抗生素基因。蓝白斑筛选在指示培养基上，未转化质粒的菌落因无抗生素抗性而不能生长，重组质粒的菌落是白色的，非重组质粒的菌落是蓝色的，以颜色不同为依据直接筛选重组克隆的方法 [2]。原理播报编辑一些载体（如PUC系列质粒）带有β-半乳糖苷酶（lacZ）N端α片段的编码区，该编码区中含有多克隆位点（MCS），可用于构建重组子 [3]。这种载体适用于仅编码β-半乳糖苷酶C端ω片段的突变宿主细胞。因此，宿主和质粒编码的片段虽都没有半乳糖苷酶活性，但它们同时存在时，α片段与ω片段可通过α-互补形成具有酶活性的β-半乳糖苷酶。这样，lacZ基因在缺少近操纵基因区段的宿主细胞与带有完整近操纵基因区段的质粒之间实现了互补。由α-互补而产生的LacZ+细菌在诱导剂IPTG（异丙基硫代半乳糖苷）的作用下，在生色底物X-Gal存在时产生蓝色菌落。而当外源DNA插入到质粒的多克隆位点后，几乎不可避免地破坏α片段的编码，使得带有重组质粒的LacZ-细菌形成白色菌落。这种重组子的筛选，称为蓝白斑筛选。蓝白斑筛选原理(8张)适用方面播报编辑设计适用于蓝白斑筛选的基因工程菌为β-半乳糖苷酶缺陷型菌株。这种宿主菌基因组中编码β-半乳糖苷酶的基因突变，造成其编码的β-半乳糖苷酶失去正常N端一个146个氨基酸的短肽（即α肽链），从而不具有生物活性，即无法作用于X-gal产生蓝色物质。用于蓝白斑筛选的载体具有一段称为lacz'的基因，lacz'中包括：一段β-半乳糖苷酶的启动子；编码α肽链的区段；一个多克隆位点（MCS）。MCS位于编码α肽链的区段中，是外源DNA的选择性插入位点。虽然上述缺陷株基因组无法单独编码有活性的β-半乳糖苷酶，但当菌体中含有带lacz'的质粒后，质粒lacz'基因编码的α肽链和菌株基因组表达的N端缺陷的β-半乳糖苷酶突变体互补，具有与完整β-半乳糖苷酶相同的作用X-gal生成蓝色物质的能力，这种现象即α-互补。操作中，添加IPTG（异丙基硫代-β-D-半乳糖苷）以激活lacz'中的β-半乳糖苷酶的启动子，在含有X-gal的固体平板培养基中菌落呈现蓝色。以上是携带空载体的菌株产生的表型。当外源DNA（即目的片段）与含lacz'的载体连接时，会插入进MCS（位于LacZ'中的多克隆位点），使α肽链读码框破坏，这种重组质粒不再表达α肽链，将它导入宿主缺陷菌株则无α互补作用，不产生活性β-半乳糖苷酶，即不可分解培养基中的X-gal产生蓝色，培养表型即呈现白色菌落。实验中，通常蓝白筛选是与抗性筛选一同使用的。含X-gal的平板培养基中同时含有一种或多种载体所携带抗性相对应的抗生素，这样，一次筛选可以判断出：未转化的菌不具有抗性，不生长；转化了空载体，即未重组质粒的菌，长成蓝色菌落；转化了重组质粒的菌，即目的重组菌，长成白色菌落。常见问题播报编辑一、没有蓝斑①载体处理的好，实验很成功，全是阳性克隆。②使用的培养基含有乳糖或葡萄糖，因为半乳糖苷酶会分解乳糖，导致不能与X-gal反应，而葡萄糖不会诱导融合基因的表达。③操作时，Amp、IPTG和X-gal其中一种或几种没涂均匀。二、没有白斑①外源目的基因没连接上，可能有外源目的基因的问题，如片段过大，或者是载体的问题。②实验操作过程中抑菌做得不好，都是杂菌。③忘记加入诱变剂IPTG或生色底物X-gal，或这两种试剂变质。④插入的外源目的基因片段较短，且插入后的基因读码框恰好和lacZ基因相吻合。三、无菌落生长①IPTG使用浓度太高，IPTG对菌体是有毒性的。②Amp使用浓度过高。③感受态细胞全部死亡。 [4]新手上路成长任务编辑入门编辑规则本人编辑我有疑问内容质疑在线客服官方贴吧意见反馈投诉建议举报不良信息未通过词条申诉投诉侵权信息封禁查询与解封©2024 Baidu 使用百度前必读 | 百科协议 | 隐私政策 | 百度百科合作平台 | 京ICP证030173号京公网安备110000020000

蓝白斑筛选的原理是什么？ - 知乎

蓝白斑筛选的原理是什么？ - 知乎首页知乎知学堂发现等你来答切换模式登录/注册生物学分子生物学细胞生物学蓝白斑筛选的原理是什么？百度上说野生型大肠杆菌能使 x-gal变成蓝色，又说单单是宿主不能使x-gal变蓝，因为没有α互补作用，而是是否矛盾？那么实验室用做转化用的大肠杆菌是…显示全部关注者80被浏览278,828关注问题写回答邀请回答好问题 3添加评论分享11 个回答默认排序知乎用户这是我在自己公众号上写的。公众号BioEngX分子克隆实验是分子生物学中常见的一个实验。感兴趣的基因插入到表达载体中，而后转化进入宿主内，但是并非所有质粒都包含目的基因，怎样筛查出包含有目的基因的菌落呢，这样的菌落也就是我们所说的阳性克隆。早期的科学家发展了蓝白斑筛选这种狂拽炫酷的东西，只需通过菌落的颜色，我们能够轻松找出阳性克隆。今天，小编就为大家简单介绍下隐含在这种酷炫技术背后的秘密。蓝白斑筛选的原理首先我们需要知道是什么让菌落显现出蓝白两色的。其实呢，白色吧，就是菌落本身的颜色，汗。是真的，看一下平板就知道了，菌落就是白色的。那蓝色呢？蓝色确实是比较神奇的。蓝色其实是β-半乳糖苷酶（β-galactosidase enzyme）分解X-gal产生的产物的颜色。X-gal？什么鬼。它的全名是5-溴-4-氯-3-吲哚-β-D-半乳糖苷（不要试图记住全名了，为了记住它，小编已经阵亡）。产物又是啥，产物是一种叫做5-溴-4-氯靛兰的蓝色东东。大肠杆菌本身是能够表达β-半乳糖苷酶的（乳酸存在的前提下），所以正常的大肠杆菌无法进行蓝白斑筛选，有乳酸存在的条件下，所有菌落都会分解X-gal产生蓝色。能够用来进行蓝白斑筛选的菌株，我们管之叫作为β-半乳糖苷酶缺陷型菌株。缺陷就意味这不正常。也就是说能够进行蓝白斑筛选的菌株内的β-半乳糖苷酶的表达是不正常的（小编你好啰嗦）。β-半乳糖苷酶可以拆分成两个部分，N端和C端。β-半乳糖苷酶缺陷型菌株的基因组中含有表达β-半乳糖苷酶C端的基因，而N端（一个146个氨基酸的短肽，即α肽链）的基因被安放到了表达的载体中。N端基因经过改造，中间插入多克隆位点。这段经过改造的N端基因被称为lacZ基因（见图1）。图1 适用于蓝白斑筛选表达载体LacZ基因是神一般的存在。神在何处，听小编慢慢道来。缺陷株基因组无法单独编码有活性的β-半乳糖苷酶，但当菌体中含有带lacZ的质粒后，质粒lacZ基因编码的α肽链（酶的N端）和菌株基因组表达的β-半乳糖苷酶的C端互补，具有与完整β-半乳糖苷酶相同的作用，具有分解X-gal生成蓝色物质的能力。这种现象也叫α-互补。操作中，添加IPTG(异丙基硫代-β-Ｄ-半乳糖苷)以激活lacZ中的β-半乳糖苷酶的启动子，在含有X-gal的固体平板培养基中菌落就会呈现蓝色。但是当MCS位点上插入DNA片段时，lacZ基因就失活了，它突变了，表达出来的东西连它的兄弟，β-半乳糖苷酶C端都不认识，没法完成α互补，β-半乳糖苷酶失活，无法分解X-gal，因此菌落就是白色的。好神奇啊？图2 蓝白斑筛选显色原理那么问题来了，β-半乳糖苷酶缺陷型菌株都有哪些？与之配套的表达质粒又有哪些呢？小编在这里简单列举几个常用的：β-半乳糖苷酶缺陷型菌株有DH5alpha、TOP10等；适用于蓝白斑筛选的载体有Puc系列（Puc18 ，Puc19）、M13mp系列等。蓝白斑筛选的不足经过上面啰哩叭嗦的解释，大家应该明白蓝白斑筛选的原理了。但是“再炫的技术，也会有自己的软肋”，这条至理名言同样适用于蓝白斑筛选(这是哪里的至理名言，小编你瞎编的吧！? 额，是的) 。蓝白斑筛选的不足之处在于，如果在表达载体内插入的基因太短同时没有破坏掉lacZ的阅读框，表达的α肽链可能还是有些活性的，菌落仍然会显蓝色。这就产生了传说中的假阴性。最后解释下开放阅读框（open reading frame）：开放阅读框是基因序列的一部分，包含一段可以编码蛋白的碱基序列。由于拥有特殊的起始密码子和直到可以从该段碱基序列产生合适大小蛋白才出现的终止密码子，该段碱基序列编码一个蛋白。（这句话完全来在百度百科，如有雷同，那也是来自百度百科）原文链接见 http://www.bioengx.com/2016/02/26/blue-white-screening/编辑于 2016-03-01 00:24赞同 25028 条评论分享收藏喜欢收起Mono生物学话题下的优秀答主关注蓝白斑筛选利用的是乳糖操纵子系统。野生大肠杆菌具有完整的乳糖操纵子系统，能把 x-gal变成蓝色。实验室用做转化用的大肠杆菌缺乏乳糖操纵子系统中的lac-Z基因。这个基因被普遍设计在了各种各样的载体上，并且在lac-Z基因编码区中间涉及了多克隆位点，以供外源基因插入。而外源基因的插入又会导致Lac-Z基因被破坏。因此，没有受到质粒转入的感受态菌会被抗生素杀死，得到了空质粒的大肠杆菌不会被杀死，而且能够将 x-gal变成蓝色。只有得到了具有插入片段的质粒的大肠杆菌不会被抗生素杀死，且不能分解X-gal，于是长出白斑。编辑于 2014-11-05 13:27赞同 918 条评论分享收藏喜欢

《微生物基础技术》蓝白斑筛选的原理是什么？ - 知乎

《微生物基础技术》蓝白斑筛选的原理是什么？ - 知乎首发于微生物基本技术切换模式写文章登录/注册《微生物基础技术》蓝白斑筛选的原理是什么？54微语者微生物发酵首先，搞清楚蓝白斑筛选的目的是什么？其次，筛选原理是什么？为什么？最后，如何应用蓝白斑筛选？一、蓝白斑筛选的目的就是为了筛选基因工程中细菌重组子。构建工程菌就是为了朝着对人类有用的方向改造，产生我们需要的酶等产物。所以要找到一种方法，筛选出这个重组子，以备后续使用。二、蓝白斑筛选的原理基于遗传特性。这是曾经看过的一篇文章，讲解清晰，但文字太多，看的有点头大，我又在原来基础上加工了一下，都是库存呀，应该相当直观。接下来我们一条一条分析：1、X-gal在β-半乳糖苷酶的作用下能够产生蓝色化合物。2、正常大肠杆菌在异乳糖存在的情况下能够分泌β-半乳糖苷酶，也能够将X-gal分解产生蓝色化合物。3、β-半乳糖苷酶缺陷型菌株β-半乳糖苷酶表达异常，不能够分泌β-半乳糖苷酶，所以不能分解X-gal。明白上述三点后，我们进一步分析。有个α-互补概念需要熟悉一下。看下图，α-互补1、β-半乳糖苷酶有一个N端，一个C端。2、N端存在表达载体中，中间插入多克隆位点后，就叫lacZ基因。我们就得到了含有lacZ基因的质粒。3、β-半乳糖苷酶缺陷型菌株的基因组中含有表达β-半乳糖苷酶C端的基因。现在应该明白了，缺陷型菌株和质粒结合就完美了。4、二者互补，具有分泌β-半乳糖苷酶的能力，能够分解X-gal产生蓝色化合物，这种现象也叫α-互补。原理基本上说完了，继续看下图。这就是构建后出现的三种情况，也就是蓝白斑筛选，我们挑选蓝色的菌落即可。有问题请留言，知无不言，及时回复，一起加油！原文http://www.bioengx.com/2016/02/26/编辑于 2024-03-05 21:14・IP 属地北京微生物微生物学微生物技术赞同添加评论分享喜欢收藏申请转载文章被以下专栏收录微生物基

蓝白斑筛选，一种很炫的东西 - 知乎

蓝白斑筛选，一种很炫的东西 - 知乎首发于BioEngX切换模式写文章登录/注册蓝白斑筛选，一种很炫的东西于浩然Protein Engineer分子克隆实验是分子生物学中常见的一个实验。感兴趣的基因插入到表达载体中，而后转化进入宿主内，但是并非所有质粒都包含目的基因，怎样筛查出包含有目的基因的菌落呢，这样的菌落也就是我们所说的阳性克隆。早期的科学家发展了蓝白斑筛选这种狂拽炫酷的东西，只需通过菌落的颜色，我们能够轻松找出阳性克隆。今天，小编就为大家简单介绍下隐含在这种酷炫技术背后的秘密。（经知乎网友@喵喵咪鸭提醒后，附上的蓝白斑筛选agar plate图片）蓝白斑筛选的原理首先我们需要知道是什么让菌落显现出蓝白两色的。其实呢，白色吧，就是菌落本身的颜色，汗。是真的，看一下平板就知道了，菌落就是白色的。那蓝色呢？蓝色确实是比较神奇的。蓝色其实是β-半乳糖苷酶（β-galactosidase enzyme）分解X-gal产生的产物的颜色。X-gal？什么鬼。它的全名是5-溴-4-氯-3-吲哚-β-D-半乳糖苷（不要试图记住全名了，为了记住它，小编已经阵亡）。产物又是啥，产物是一种叫做5-溴-4-氯靛兰的蓝色东东。大肠杆菌本身是能够表达β-半乳糖苷酶的（乳酸存在的前提下），所以正常的大肠杆菌无法进行蓝白斑筛选，有乳酸存在的条件下，所有菌落都会分解X-gal产生蓝色。能够用来进行蓝白斑筛选的菌株，我们管之叫作为β-半乳糖苷酶缺陷型菌株。缺陷就意味这不正常。也就是说能够进行蓝白斑筛选的菌株内的β-半乳糖苷酶的表达是不正常的（小编你好啰嗦）。β-半乳糖苷酶可以拆分成两个部分，N端和C端。β-半乳糖苷酶缺陷型菌株的基因组中含有表达β-半乳糖苷酶C端的基因，而N端（一个146个氨基酸的短肽，即α肽链）的基因被安放到了表达的载体中。N端基因经过改造，中间插入多克隆位点。这段经过改造的N端基因被称为lacZ基因（见图1）。图1 适用于蓝白斑筛选表达载体LacZ基因是神一般的存在。神在何处，听小编慢慢道来。缺陷株基因组无法单独编码有活性的β-半乳糖苷酶，但当菌体中含有带lacZ的质粒后，质粒lacZ基因编码的α肽链（酶的N端）和菌株基因组表达的β-半乳糖苷酶的C端互补，具有与完整β-半乳糖苷酶相同的作用，具有分解X-gal生成蓝色物质的能力。这种现象也叫α-互补。操作中，添加IPTG(异丙基硫代-β-Ｄ-半乳糖苷)以激活lacZ中的β-半乳糖苷酶的启动子，在含有X-gal的固体平板培养基中菌落就会呈现蓝色。但是当MCS位点上插入DNA片段时，lacZ基因就失活了，它突变了，表达出来的东西连它的兄弟，β-半乳糖苷酶C端都不认识，没法完成α互补，β-半乳糖苷酶失活，无法分解X-gal，因此菌落就是白色的。好神奇啊？那么有看官要问了，β-半乳糖苷酶缺陷型菌株都有哪些？与之配套的表达质粒又有哪些呢？小编在这里简单列举几个常用的：β-半乳糖苷酶缺陷型菌株有DH5alpha、TOP10等；适用于蓝白斑筛选的载体有Puc系列（Puc18 ，Puc19）、M13mp系列等。蓝白斑筛选的不足经过上面啰哩叭嗦的解释，大家应该明白蓝白斑筛选的原理了。但是“再炫的技术，也会有自己的软肋”，这条至理名言同样适用于蓝白斑筛选 (这是哪里的至理名言，小编你瞎编的吧！? 额，是的)。蓝白斑筛选的不足之处在于，如果在表达载体内插入的基因太短同时没有破坏掉lacZ的阅读框，表达的α肽链可能还是有些活性的，菌落仍然会显蓝色。这就产生了传说中的假阴性。最后解释下开放阅读框（open reading frame）：开放阅读框是基因序列的一部分，包含一段可以编码蛋白的碱基序列。由于拥有特殊的起始密码子和直到可以从该段碱基序列产生合适大小蛋白才出现的终止密码子，该段碱基序列编码一个蛋白。（这句话完全来在百度百科，如有雷同，那也是来自百度百科）编辑于 2017-04-03 16:17分子生物学生物技术基因工程赞同 476 条评论分享喜欢收藏申请转载文章被以下专栏收录BioEngX活跃在移动端的无国界

Blue-White Screening & Protocols for Colony Selection

-White Screening & Protocols for Colony SelectionPHENProductsApplicationsServicesDocumentsSupportAnalytical ChemistryCell Culture & AnalysisChemistry & BiochemicalsClinical & DiagnosticsFiltrationGreener Alternative ProductsIndustrial MicrobiologyLabwareMaterials ScienceMolecular Biology & Functional GenomicsmRNA Development & ManufacturingPharma & Biopharma ManufacturingProtein BiologyWater PurificationAnalytical ChemistryCell Culture & AnalysisChemistry & Synthesis Clinical & DiagnosticsEnvironmental & Cannabis TestingFood & Beverage Testing & ManufacturingGenomicsMaterials Science & EngineeringMicrobiological TestingmRNA Development & ManufacturingPharma & Biopharma ManufacturingProtein BiologyResearch & Disease AreasContract ManufacturingContract TestingCustom ProductsDigital Solutions for Life ScienceIVD Development & ManufacturingmRNA Development & ManufacturingProduct ServicesSupportSafety Data Sheets (SDS)Certificates of Analysis (COA)Certificates of Origin (COO)Certificates of Quality (COQ)Customer SupportContact UsFAQSafety Data Sheets (SDS)Certificates (COA/COO)Quality & RegulatoryCalculators & AppsWebinarsHomeCloning & ExpressionBlue-White Screening & Protocols for Colony SelectionBlue-White Screening & Protocols for Colony Selection Identification of Recombinant Bacteria

Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria. It relies on the activity of β-galactosidase, an enzyme occurring in E. coli, which cleaves lactose into glucose and galactose.

Disrupting the LacZ Gene

The presence of lactose in the surrounding environment triggers the lacZ operon in E. coli. The operon activity results in the production of β-galactoisdase enzyme that metabolizes the lactose. Most plasmid vectors carry a short segment of lacZ gene that contains coding information for the first 146 amino acids of β-galactosisdase. The host E. coli strains used are competent cells containing lacZΔM15 deletion mutation. When the plasmid vector is taken up by such cells, due to α-complementation process, a functional β-galatosidase enzyme is produced.

The plasmid vectors used in cloning are manipulated in such a way that this α-complementation process serves as a marker for recombination. A multiple cloning site (MCS) is present within the lacZ sequence in the plasmid vector. This sequence can be nicked by restriction enzymes to insert the foreign DNA. When a plasmid vector containing foreign DNA is taken up by the host E. coli, the α-complementation does not occur, therefore, a functional β-galactosidase enzyme is not produced. If the foreign DNA is not inserted into the vector or if it is inserted at a location other than MCS, the lacZ gene in the plasmid vector complements the lacZ deletion mutation in the host E. coli producing a functional enzyme.

How Does Blue White Screening Work?

For screening the clones containing recombinant DNA, a chromogenic substrate known as X-gal is added to the agar plate. If β-galactosidase is produced, X-gal is hydrolyzed to form 5-bromo-4-chloro-indoxyl, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5’-dibromo-4,4’-dichloro-indigo. The colonies formed by non-recombinant cells, therefore appear blue in color while the recombinant ones appear white. The desired recombinant colonies can be easily picked and cultured.

Isopropyl β-D-1-thiogalactopyranoside (IPTG) is used along with X-gal for blue-white screening. IPTG is a non-metabolizable analog of galactose that induces the expression of lacZ gene. It should be noted that IPTG is not a substrate for β-galactosidase but only an inducer. For visual screening purposes, chromogenic substrate like X-gal is required.

Figure 1.A schematic representation of a typical plasmid vector that can be used for blue-white screening.Figure 2.A schematic representation of a typical blue-white screening procedure.Blue-White Screening Products (Materials)

Protocols for Blue-White Screening

The complete protocol of blue-white screening includes 3 important steps:

Ligation: ligation of foreign DNA into MCS of the plasmid vector

Transformation: introduction of plasmid vector with foreign DNA insert into competent E. coli

Screening: blue-white screening to identify recombinant bacterial colonies

Ligation

We offer the following ready-to-use expression vectors that can be used for stable as well as transient expression systems. These FLAG-fusion constructs have origins of replication for propagation in both bacterial and mammalian cells.

Materials required

The following is the reaction setup for ligation

Add all the above components into a clean reaction tube.

Incubate for 30 minutes at 25 °C (can be performed in a thermo cycler).

Purify DNA using PCR clean-up column and elute in approximately 50 µL.

Transform 0.1-10 ng of the ligation product into chemical or electrocompetent cells that are compatible with the vector.

Transformation

High quality plasmid is essential for the transformation procedure. The following kits isolate and purify high quality plasmid suitable for transformation.

The detailed protocol for transformation using chemical or electrocompetent cells can be found here.

Screening

We offer a range of chromogenic substrates that aid screening of recombinant bacteria. Some products may be used to spread on LB agar plates (screening protocol 1), while the others are incorporated into the microbial medium (screening protocol 2). The products are used along with IPTG wherever required. The protocols for both the procedures are given below.

Screening protocol 1 (applicable for product numbers B6650, 16658, B2904, B3928, 16669, B8931)

Spread 40 µL or appropriate amount of stock solution of chromogenic substrate and 10 µL of IPTG solution on LB agar plates using a sterile spreader (addition of IPTG when using product number B3928 is not required as it already contains IPTG).

The plates should include those with appropriate antibiotic and without antibiotic as controls.

Leave the plates to dry in laminar flow chamber with lids slightly open.

Spread 10-100 µL of transformed E. coli cells onto the LB agar plates using sterile spreader.

Incubate the plates at 37 °C for 24-48 hours.

Blue and white colonies appear on the agar surface. Select the recombinant cells in the white colonies to culture.

Screening protocol 2 (applicable for product numbers S7313, S9811, C4478)

Prepare LB agar by weighing appropriate powder medium, agar and water in a sterile flask. Alternatively, weigh appropriate amount of C4478 and add to water in a sterile flask.

Add 300 mg/L of product numbers S7313 and S9811, and 500 mg/L of ferric ammonium citrate (product number F5879) to the medium before autoclaving. Avoid this step if C4478 is being used.

Autoclave the medium and cool just enough to be able to handle the flask.

Add appropriate concentration of selected antibiotic to the medium.

Pour approximately 25-30 mL of the LB agar into sterile plates and allow to set with lids slightly open.

Spread 10-100 µL of transformed E. coli cells onto the LB agar plates using sterile spreader.

Incubate the plates at 37 °C for 24-48 hours.

Blue and white colonies appear on the agar surface. Select the recombinant cells in the white colonies to culture.

Figure 3.Blue-white color selection of recombinant bacteria using X-gal.Limitations of blue-white screening

The blue-white technique is only a screening procedure; it is not a selection technique.

The lacZ gene in the vector may sometimes be non-functional and may not produce β-galactosidase. The resulting colony will not be recombinant but will appear white.

Even if a small sequence of foreign DNA may be inserted into MCS and change the reading frame of lacZ gene. This results in false positive white colonies.

Small inserts within the reading frame of lacZ may produce ambiguous light blue colonies as β-galactosidase is only partially inactivated.

References1. Sambrook J, Fritsch E, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. 1. New York: Cold Spring Harbor Laboratory Press.2. Padmanabhan S, Banerjee S, Mandi N. Screening of Bacterial Recombinants: Strategies and Preventing False Positives. https://doi.org/10.5772/22140 Related ArticlesIntroduction to Cell TransfectionReverse Transfection of Plasmid DNA3xFLAG® System Expression Vectors for Ultra-Sensitive Detection of Recombinant ProteinsBacterial Transformation ProtocolsGenotypes, Phenotypes and MarkersRestriction Enzyme Cloning GlossaryHow Transfection WorksIntroduction to Yeast TransformationView MoreRelated Product CategoriesDetection Substrates & EnzymesMolecular Cloning & Protein ExpressionDetergents - Anionic, Cationic, Zwitterionic, Anti-foamingCarbohydrates for Biochemical ResearchPlasmid DNA Purification TopSign In To ContinueTo continue reading please sign in or create an account.Sign InDon't Have An Account?Regis

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Home›Life Sciences›Molecular Cloning Essentials›Molecular Biology Education›Molecular Cloning Education›Traditional Cloning Education›Traditional Cloning BasicsTraditional Cloning BasicsSee Navigation‹Traditional Cloning EducationTraditional Cloning Basics

›Cloning Troubleshooting Guide

›Common Cloning Applications and Strategies

›Traditional cloning relies on recombinant DNA methods that begin with preparing a vector to receive an insert DNA by digesting each with restriction enzymes. The digested fragments are then spliced together by an enzyme called ligase, in a process known as ligation, to form a new vector capable of expressing a gene of interest. This may be the simplest and oldest technique for traditional cloning and laid the foundation for researchers to develop novel cloning methods such as TA cloning, TOPO cloning, PCR cloning, ligation-independent cloning, and gene assembly that exploit unique characteristics of other modifying enzymes.A general workflow for traditional cloning includes the following steps (Figure 1):1. Vector preparation2. Insert preparation3. Ligation4. Transformation5. Colony screeningFigure 1. Traditional cloning workflow.

Vector preparation

Vectors used in traditional cloning methods are based on plasmids, which are double-stranded, circular DNAs that replicate inside bacteria independently of the genomic DNA. All cloning vectors based on plasmids contain a number of crucial elements, including a bacterial origin of replication to efficiently propagate within the bacterial host cell; single restriction enzyme site(s) or, more commonly, a multiple cloning site (MCS) that contains a number of restriction enzyme sites to allow ready addition of an insert of interest; and a marker (e.g., antibiotic resistance) to select for bacteria after successful uptake of the vector.In some vectors, the MCS is located within a gene that serves as a marker and permits screening for clones into which the insert has been spliced successfully. For instance, the pUC18 vector expresses the lacZα gene encoding the alpha peptide of beta-galactosidase which, in combination with X-gal, allows color selection of bacterial colonies formed after cloning (learn more about blue/white selection in colony screening).Figure 2. Map of pUC18 with its MCS.The first step in preparing the vector for traditional cloning is to create an insertion site by restriction digestion. The choice of restriction enzymes depends upon the presence and location of their recognition sequences on the vector and the insert, and their compatibility for ligation. Vector restriction sites can be found on the vector map, or can be mapped using free online tools such as RestrictionMapper. The MCS, if available, is often the first choice for insertion, as the region is specifically designed for cloning.After restriction digestion, dephosphorylation of the vector may be necessary to prevent self-ligation, especially if the resulting ends of vector digestion are compatible or blunt. During dephosphorylation, the enzyme alkaline phosphatase removes the 5′ phosphate groups at the ends. This prevents vector self-ligation because the enzyme ligase requires both a 5′ phosphate and a 3′ OH to join the two ends in recircularization of the vector (see Ligation). (App note: Dephosphorylation)Dephosphorylation of the vector is important to reduce background and favor insertion of the desired fragment into the vector. Both self-ligated vector molecules and insert-carrying vector molecules can be taken up by the bacteria during transformation and will confer the same antibiotic resistance to those cells (see Colony screening and Figure 6). This will create a higher background of undesirable colonies if the vector is not dephosphorylated.Blunting of the vector ends may be required, depending upon the restriction enzymes used. Purification of the desired fragments is also recommended for successful ligation.(Top)

Insert preparation

The source of the insert for cloning may be genomic DNA, a portion of another plasmid, or a linear DNA fragment. Regardless of the type of source DNA, a common first step in preparation of the insert is to perform restriction digestion to generate compatible ends for subsequent splicing into the vector.As with vector preparation, restriction enzymes that are suitable for cloning of the insert into the vector are selected. One of the most popular strategies is to perform double digests of both the insert and vector for directional cloning. In the following example (Figure 3A), two enzymes that generate non-compatible ends (EcoRI and KpnI) are used. Since vector and insert ends can join in only one orientation due to compatibility (EcoRI with EcoRI, KpnI with KpnI), this approach allows the insert to be cloned directionally. (App note: Directional cloning).Figure 3. Common restriction enzyme cloning strategies.(A) Double digestion of the vector and the insert (e.g., EcoRI and KpnI) for directional cloning. (B) Single digestion of the vector and the insert with two separate restriction enzymes (e.g., BamHI and PstI), followed by blunt-end digestion or polishing. (C) Single digestion of the vector and the insert with the same restriction enzyme (e.g., BamHI) for cloning. (D) Single digestion of the vector and the insert with two restriction enzymes with compatible ends (e.g., BamHI and BglII) for cloning.When performing double digestion, it is crucial that the reaction buffer and conditions are optimal for both enzymes; therefore, manufacturers’ recommendations for double digest reaction setups should be followed closely to ensure success. Some restriction enzymes are designed for complete digestion with multiple enzymes in a single buffer, enabling simplicity and time saving.In instances where suitable restriction enzymes are not available, the DNA ends created by the chosen restriction enzymes may be blunted (or “polished”) for cloning. Blunting will alter the original sequences around the DNA ends; this could cause a frameshift in the gene translation region or disruption within a gene regulatory region. In addition, ligation of DNA with blunt ends is typically more challenging and less efficient than with cohesive (“sticky”) ends (Figure 3B).In some instances, a single restriction enzyme may be chosen that cuts both the insert and vector DNA, generating complementary ends for ligation (Figure 3C). This method is commonly used in genomic DNA cloning.In situations when it is not possible to use a single restriction enzyme, a pair of enzymes that have different recognition sequences but generate compatible overhangs can be considered as an alternative. For example, BamHI recognizes 5′-G↓GATCC-3′ and BglII recognizes 5′-A↓GATCC-3′; both generate 5′-GATC overhangs that can be joined in a ligation reaction. Note, however, that neither recognition site is restored after ligation in this case (Figure 3D).Commonly used enzymes for generating blunt ends are the large (“Klenow”) fragment of DNA polymerase I, and T4 DNA polymerase. The choice of polymerase depends on whether the restriction enzyme generates a 3′ or 5′ overhang. In the case of 3′ overhangs (e.g., those generated by KpnI), T4 DNA polymerase is preferred because it has a stronger 3′ to 5′ exonuclease activity than does Klenow. In this scenario, the 3′ overhang is digested or “chewed back” by the T4 DNA Polymerase. For 5′ overhangs (e.g., those generated by EcoRI), either Klenow or T4 DNA Polymerase can be used to fill in the overhangs through their 5′ to 3′ polymerase activity. In a few instances, mung bean nuclease or S1 nuclease, added in excess, can be used to trim single-stranded DNA overhangs through their 5′ to 3′ exonuclease activities on single-stranded DNA (Figure 4).Figure 4. Enzymatic digestion to produce blunt ends from overhangs created by restriction digestion.After restriction digestion of the insert and the vector (and subsequent blunting and dephosphorylation, if performed), the desired fragments can be purified by running the samples on an agarose gel and excising the fragments of interest. Gel electrophoresis also removes enzymes and salts that were present in the digestion reactions. Gel purification kits are commercially available for efficient workflow, reliable results, and high yields. The kits are based on procedures that use a chaotropic reagent and heat to liquefy the gel, after which the fragments are purified using silica columns or magnetic beads. For gentle and efficient recovery of long DNA fragments (e.g., >10 kb), low melting gel, in combination with the enzyme agarase, which breaks down the agarose matrix, can be used. The traditional method for nucleic acid recovery from the solubilized gel is phenol/chloroform extraction, followed by ethanol precipitation of the fragments. The phenol/chloroform extraction may, however, result in lower yield and carryover of phenol that can affect downstream experiments. Extracted DNA should be highly pure for successful ligation. The simplest method to assess purity is to measure its absorbance: pure DNA has an A260/A280 ratio of >1.8 and an A260/A230 ratio of approximately 2.0.xWhat is a purity ratio in measuring DNA absorbance?(Top)

Ligation

Once the fragments of interest are obtained, a ligation reaction can be set up to join the insert and the vector. The most common enzyme used for ligation is T4 DNA ligase, which links DNA ends between 5′ phosphate and 3′ OH groups. The T4 DNA ligase reaction requires ATP, DTT, and Mg2+, which are generally supplied in the reaction buffer (Figure 5). To improve the outcome of ligation, a general recommendation is to set up multiple reactions with varying insert:vector molar ratios, typically in the range of 1:1 to 5:1. (Download the CloningBench app to access convenient tools and calculators, including the Vector to Insert Molar Ratios Calculator.) For less efficient ligations, as with DNA fragments with blunt ends, the addition of inert macromolecules like polyethylene glycol (PEG) is often recommended to increase the effective concentration of reaction components and thus improve the ligation efficiency.Figure 5. T4 DNA ligase reaction.Reaction temperatures may range from 14°C to 25°C (room temperature), and reaction times from 10 minutes to 16 hours (or overnight), depending on the type of DNA fragments and desired yields. In general, a higher reaction temperature requires less time but may produce a lower yield. Some commercially available ligation kits are designed to attain complete ligation in 15 minutes at room temperature. (App note: Ligation).The ligated mixture may be used directly in transformation of chemically competent cells but may require purification prior to transformation of electrocompetent cells. If PEG was used in the ligation reaction, heat inactivation of the ligase is not recommended after the reaction, since this can reduce transformation efficiency.(Top)

Transformation

Transformation is a naturally occurring process in which bacterial cells take up foreign DNA at a low frequency. In molecular biology applications, this process is enhanced and exploited to propagate plasmids inside bacteria that have been made “competent” (porous) for DNA uptake.Competent cells are commercially available for efficient and reliable transformation. The most common approach to prepare bacteria to be competent for transformation is to treat log-phase bacterial cells with calcium chloride. When the chemically competent cells are mixed with the DNA from the ligation reaction and then heat-shocked at 42°C, some of the DNA is absorbed by the bacterial cells, where it begins to replicate.xBasics of competent cell transformationDifferent strains of competent cells are available, and the choice is based on experimental goals and downstream applications. For instance, to perform “blue/white” screening, a bacterial strain with a lacZ mutation (lacZΔM15) must be chosen. If the experiment calls for digestion with methylation-sensitive restriction enzymes, the plasmid should be propagated in a dcm–/dam– bacterial strain. For protein expression, the strain should accommodate mRNA stability and translation, as well as high induction of the recombinant protein’s expression. (Learn more: Competent cell selection by applications).In addition, transformation efficiency of the competent cells is an important consideration. Manufacturers provide the transformation efficiency of competent cells in colony-forming units per microgram of DNA (CFU/µg), generally ranging from 1 x 106 to 1 x 109 CFU/µg. In more difficult ligation and cloning strategies, choosing cells with the highest transformation efficiencies can greatly improve the likelihood of obtaining the desired clones.Another method to transform bacterial cells is electroporation. In this technique, electrocompetent bacterial cells and ligated plasmids are treated with an electrical current that creates transient pores in the bacterial cell membrane for DNA uptake.Transformed bacteria (after heat shock or electroporation) are then plated on an agar plate with an appropriate antibiotic, and screened (by blue-white screening or another method) for colonies that carry the desired plasmid with insert.(Top)

Colony screening

The transformation reaction contains a mix of cells with no vector, the vector with no insert, the insert alone, and the successfully ligated vector and insert. Bacteria without the vector lack the antibiotic resistance gene and will not grow, whereas bacteria transformed with the vector (with or without the insert) survive due to the expressed antibiotic resistance gene (Figure 6). Thus, the antibiotic resistance allows selection for uptake of an intact plasmid.Figure 6. Mixture of bacteria after transformation and their phenotypes (growth/no growth/blue-white) on an antibiotic selection media plate. The ligation mixture may include failed products (unligated insert, unligated vector and the no-insert/empty vector), as well as desired/undesired insert-carrying vectors. The undesired insert may harbor an incorrect, mutated or truncated sequence.To identify whether the transformed colonies contain an insert, a number of methods can be employed, of which the most common are “blue/white” screening and positive selection. Blue/white screening relies on transforming a bacterial strain that expresses a mutant lacZ gene (lacZΔM15), which can be complemented with the alpha peptide of beta-galactosidase, encoded on the vector (alpha complementation). Transformed cells are plated on a growth medium that includes a transcriptional inducer for lacZ expression, IPTG, and a chromogenic substrate of LacZ, X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). In blue/white screening, LacZ will hydrolyze the X-gal, producing a blue dye and hence a blue colony. When a DNA insert disrupts the vector-encoded lacZα gene, no functional LacZ is formed, and transformed colonies are white (Figure 7).Figure 7. Color formation by beta-galactosidase (β-gal) activity on X-gal, and its application in blue/white screening.Another popular screening approach is positive selection, whereby a gene lethal to the bacterial host is located in the MCS of the vector. Successful ligation of an insert into the lethal gene in the MCS prevents its expression, allowing only transformed cells with insert-carrying vectors to survive.To more specifically identify or characterize the insert, transformed colonies must be further analyzed, as the results of blue/white screening and positive selection can only provide information about whether an insert is present or not. One basic approach is to perform restriction digestion of the vector extracted from the positive or white colonies and examine the resulting banding patterns from gel electrophoresis. Restriction enzymes must be carefully chosen and can be used to confirm the size and orientation of the insert.The presence of the DNA insert can also be determined by a method called colony PCR, in which a small portion of the colonies is analyzed by PCR. This method requires PCR primers that are specific to the insert, to the flanking vector sequences, or both, to detect the insert. To determine the orientation of the insert, a set of primers that can detect the vector and the insert in a single reaction can be designed (Figure 8). (App note: Colony PCR).Figure 8. Potential primer design approaches in colony PCR screening. The insert-specific primers are depicted as red arrows and the vector-specific primers as green. A schematic gel electrophoresis analysis of colony PCR products from the empty vector (labeled “1”) and the insert-carrying vectors (labeled “2” and “3”) is shown.The most definitive way to identify the insert is Sanger sequencing (also known as dideoxy sequencing). While this is a good way to confirm the presence and precise sequence of the insert, this approach may be time-consuming and cost-prohibitive, depending upon the number of colonies to be screened.Once clones with the correct insert are identified, they are ready for downstream experiments.(Top)

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Learn moreRestriction enzymesBacterial transformationPCRGel electrophoresisRelated productsRestriction enzymesDNA modification enzymesCompetent cellsAgarose gel electrophoresisAgarose gel extraction kitsPCR enzymesShare For Research Use Only. Not for use in diagnostic procedures.

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X-Gal的作用机理及IPTG-X-gal平板制作方法 - 实验方法 - 丁香通

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首页实验方法遗传学实验技术经典遗传学实验 X-Gal的作用机理及IPTG-X-gal平板制作方法

X-Gal的作用机理及IPTG-X-gal平板制作方法

关键词：作用机理

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【原创】蓝白斑筛选 - 实验方法 - 丁香通

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首页实验方法 PCR技术其他PCR技术【原创】蓝白斑筛选

【原创】蓝白斑筛选

关键词：蓝白斑筛选

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实验中，通常蓝白筛选是与抗性筛选一同使用的。含X-gal的平板培养基中同时含有一种或多种载体所携带抗性相对应的抗生素，这样，一次筛选可以判断出：未转化的菌不具有抗性，不生长；转化了空载体，即未重组质粒的菌，长成蓝色菌落；转化了重组质粒的菌，即目的重组菌，长成白色菌落。

目前很多实验室省去蓝白斑筛选的步骤，造成IPTG和X-Gal滞销

主要是蓝白斑筛选存在假阳性的情况，IPTG和X-Gal的购买、配制、涂布也增加了实验的步骤

针对这个情况，我们现在推出一个新品相：Ready to use LB Agar，这种粉末里面已经添加了IPTG和X-Gal，同LB Agar的使用方法完全相同，称量、溶解，高温高压灭菌，使用前加入相应抗生素即可

如单独购买IPTG和X-Gal，则配制好的X-Gal需要避光保存

Ready to use LB Agar(含X-gal,IPTG) 1.(LB Broth加上Agar就是LB Agar)MDBio 的Ready to use LB Agar已添加了X-Gal和IPTG。2.其中使用的Agar强度可达1500（浓度1.5%）,溶解澄清度清澈,经过121度高温高压灭菌不失活。3.同一般LB Agar的使用方法,可直接称量、溶解、灭菌,但省去了自行涂布IPTG和X-Gal的麻烦。4. 在蓝白斑筛选实验中，能够使得菌落清晰地显示蓝斑或者白斑，颜色对比明显，能够清晰辨别出目的菌落。公司简介青岛生工生物科技有限公司成立于2002年，是由生工有限公司（台湾）在中国大陆投资兴创的台商独资企业。生工有限公司（台湾）成立于1996年，是台湾最大自有品牌生物试剂服务销售公司，主要生产开发分子生物化学试剂、PCR相关产品、抗生素类、各式抗体、塑料耗材等，另外，公司还提供DNA合成及分子生物学研究领域的相关技术服务。

青岛生工生物科技有限公司注册在美丽的沿海城市青岛，位于青岛高新技术开发区内，是由一群热情于工作的伙伴所组成，成员们多数来自于国内生命科学领域的专业人才，并经过严格的训练，对于工作始终保持热情、积极、专业及绝对负责的精神。精湛的生物专业技术，加之丰富的试剂行业经验，为公司传承发展奠定了良好的基石。

MDBio 秉承对客户负责的态度，在保证品质、品管严控的坚持理念下，深获市场客户的信赖与赞许。青岛生工生物科技有限公司近年已在国内几个大城市建立了代理及分销通路，如杭州、广州、南京、济南、泰安、天津、武汉、云南、西安、兰州、重庆、成都、北京、上海、厦门、哈尔滨等等。

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