感受态细胞制作和转化效率的研究

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天津农学院
毕业论文
中文题目:感受态细胞制作和转化效率的研究英文题目:Establishment of Competent Cells and Analysis of Transformation Efficiency
学生姓名于晓猛
系别动物科学系
专业班级2010 级动物医学专升本班
指导教师李吉霞
成绩评定
2012年6月
目录
1 前言 (1)
2 材料与方法 (2)
2.1 材料与仪器 (2)
2.1.1 菌种及质粒 (2)
2.1.2 培养基 (2)
2.1.3 仪器设备 (2)
2.2方法 (2)
2.2.1制备感受态细菌 (2)
2.2.2 DNA重组子的转化感受态大肠杆菌 (3)
2.2.3质粒DNA提取 (5)
3结果 (8)
3.1 感受态细菌转化效率观察 (8)
3.2 质粒提取结果观察 (10)
4 讨论 (11)
4.1 DNA 重组受体系统 (11)
4.2感受态细胞的制备 (11)
4.3 质粒转化效率 (12)
参考文献 (13)
致谢 (14)
附录1:外文文献原文 (15)
附录2:外文文献原文译文 (20)
摘要
质粒转化进入大肠杆菌感受态细胞是分子克隆的关键步骤,是基因克隆以及DNA文库构建等研究中频繁使用的一项重要的常规操作。

本研究通过复苏和扩增DH5α细菌,每50ml菌液用10ml预冷的0.1mol/L的CaCl2重悬,冰浴30min。

于4℃,离心回收。

每50ml初始培养物用2ml用冰浴的0.1mol/L的CaCl2重悬,100ul/份,-4℃放置24 h。

转化流程为:100ul感受态DH-5α+ 5ul PCI-Neo-hTERT冰上放置30min,42 ℃,热激90s,冰浴2min,加入400ul LB培养基,于37℃,50r/min振摇45min,之后取200ul涂布于含Amp琼脂板,37℃倒置培养12~16h。

次日观察,并随机选择阳性克隆,用LB液体培养基进行扩增,然后按照质粒提取试剂盒操作说明提取质粒,检测转化效率。

结果表明,通过上述方案可以得到活力较好的感受态细菌,经质粒转化后,效率较高,可以应用于载体的转化。

关键词:感受态细胞:质粒:转化
ABSTRACT
It is a key step for Plasmid transformed into Escherichia coli cells in the molecular cloning. The important routine operations are frequently used in the study of gene cloning and DNA library construction. In this study, recovery and amplification of DH5α bacteria, per 50 ml bacterium fluid with 10 ml precooling 0.1 mol/L of CaCl2 resuspended ice-bath 30min. In 4℃,centrifugal recovery. Initial culture of each 50ml ice bath 0.1mol / L CaCl2 resuspended with 2ml. 100ul/part,The transformation flows are as follows: Firstly, the operator puts the100ul competent of DH-5α and 5ul PCI-Neo-hTERT on the ice for 30 minutes, keeps the temperature at 42 degrees, heats it for 90 seconds and ice-bathes it for 2minutes, then adds 400 ul LB culture and shakes out it at the speed of 50 rounds per minute for 45 minutes at the temperature of 37 degrees. Secondly, the operator takes out 200ul on the agar plate with Amp, then converts and cultures it for 12 to 16 hours at the temperature of 37 degrees. Then next day, the operator observes it, chooses the positive cloning at random and uses the LB liquid culture to expend it. Then the operator extracts the plasmid according to the instruction of the Plasmid Extraction Kit and detects the transformation efficiency.It turned out that not only by using above methods we can get many more dynamic competent cells, but also shows a higher efficiency which would applied to vector conversion.
Key words: Competent Cells; Plasmid; Transformation
感受态细胞制作和转化效率的研究
于晓猛
(天津农学院动物科学系)
1 前言
分子生物学实验中,DNA重组技术和外源基因的表达是最为常用的研究手段之一[1]。

而体外构建的DNA重组子必须导入合适的受体细胞,才可以复制、增殖和表达。

载体与外源目的基因构成的重组载体可以通过转化直接导入受体细胞,从而实现基因在异源细胞的表达。

感受态细胞的制备是实现上述目标的一个重要环节,其制备质量的好坏直接影响后续工作的进行。

目前人们已经建立很多种感受态细胞的制备方法,最早的大肠杆菌感受态细胞制备的方法是Cohen[2]于1972年建立的。

该方法的主要原理是细菌通过冰冷的CaCl2 低渗溶液处理后,加入待转化质粒DNA,经42℃短时间热冲击,细菌细胞可增加对该质粒DNA的摄入。

这一方法成为目前实验室制备感受态细胞的常规方法。

电转化法[3]是另外一种常用的细胞转化方法,其主要特征为速度快而且有很高的转化效率,每微克质粒DNA转化菌落数可高达109~1010个,但这一方法要求昂贵的设备,技术性较强。

1990年Nishimura A报道的一种镁盐法(Nishimura)制备感受态细胞的方法,该方法制备的感受态细胞效价高可达到108,但该方法操作仍显复杂。

1989年Chung 等报道转化效率比CaCl2法要高的TSS法,而且不需要热休克。

同时还采用与TSS法操作完全相同,仅用等摩尔浓度MgSO4替换MgCl2的NewTSS法。

周国林等研究结果表明上述4种方法中TSS转化效率最高,其次为CaCl2法,再次为Nishimura法,最次为NewTSS法[4]TSS法对细胞的毒性较小,无需加入其他成分,转化方便,效率高,与其他方法相比便宜可靠,并可在-70℃下长期保存。

Tsen 等发现大肠杆菌的某些菌株可以自然地在细胞质中与细胞外质粒低频率结合。

Chen 等提出一种方便快捷的质粒转化大肠杆菌的方法, 它几乎与传统的钙转化方法有相同的转化效率, 而且这种方法大约只需2min.基于这种方法, 建立了一个利用大肠杆菌感受态细胞转化质粒的系统, 感受态细胞和质粒能长期储存, 而且便于以后实验的扩增和利用。

一般实验室没有电穿孔技术所需要的特殊仪器,而利用氯化钙法将质粒重组入大肠杆菌细胞,操作方便、应用广泛,但是氯化钙法在实际操作中的转化效率常常不能完全满足试验的要求。

目前,实验室中常用的感受态细胞多为公司直接提供的,其价格昂贵,不适于普通基因克隆操作中的频繁
使用[5]。

因此,对感受态细胞制备方法进行合理优化是提高转化效率的关键。

2 材料与方法
2.1 材料与仪器
2.1.1 菌种及质粒
PCI-Neo-hTERT质粒、大肠杆菌DH5α由天津农学院动物科学系分子育种中心保存;胰蛋白胨、酵母提取物、氨苄青霉素和小量质粒提取试剂盒等购自北京索来宝科技有限公司,DMarker III 购自上海拜利生物。

2.1.2 培养基
LB固体培养基的配方:胰蛋白胨(Tryptone) 10g/L,酵母提取物(Yeast extract) 5g/L,氯化钠(NaCl) 10g/L,琼脂粉15g/L。

配制100ml的LB培养基加入1.5g琼脂粉,高压灭菌后,将融化的LB固体培养基置与55℃的水浴中,待培养基温度降到55 ℃时(手可触摸)加入抗生素,以免温度过高导致抗生素失效,并充分摇匀。

大约10 mL倒1个板子。

培养基倒入培养皿后,打开盖子,在紫外下照10-15 min。

用封口胶封边,并倒置放于4℃保存备用。

LB液体培养基的配方:胰化蛋白胨 10g,酵母提取物 5g,NaCl 10g。

配置100 mL,高压灭菌后备用。

2.1.3 仪器设备
恒温水浴箱(常州市国立设备实验研究中心,HH-S);超净工作台(上海博讯实业有限公司医疗设备厂,SW-CJ-LF);高速冷冻离心机(Centrifuge,5424);恒温摇床(哈尔滨东联生化仪器有限公司,HZQ-F100);生化培养箱(SANYO,MCO-18AIC)。

2.2方法
2.2.1制备感受态细菌
以1:100的比例吸取过夜菌液(250ul)加入25ml LB液体培养基中,37℃,200r/min振荡培养2-3h。

将25ml菌液移至预冷的50ml聚丙烯离心管中,在冰上放置30min,使培养物冷却到0 ℃。

于4℃,以4000rpm离心10min,回收细胞。

倒出培养液,将管倒置1min(于滤纸/吸水纸上),使最后残留的痕量培养液流尽。

每50ml菌液用10ml预冷的0.1mol/L的CaCl2,重悬每份沉淀,放置于冰浴上30min。

于4℃,以4000 rpm 离心10min,回收细胞。

倒出培养液,将管倒
置1min(于滤纸/吸水纸上),使最后残留的痕量培养液流尽。

每50ml初始培养物用2ml 用冰预冷的0.1mol/L的CaCl2(含15%甘油)重悬每份细胞沉淀。

在冰上将细胞分装成小份,100ul/份,-4℃放置24 h。

(见图1-图2)
图1 感受态制备中的冰浴
2.2.2 DNA重组子的转化感受态大肠杆菌
实验流程如下所示。

100ul 感受态细胞DH-5α+ 5ul PCI-Neo-hTERT(如图2)───→轻轻旋转混合───→冰上放置30min───→42 ℃,热激90S(如图3)───→冰浴2min───→400ul LB培养基───→37℃, 50r/min振摇45min───→取200ul涂布于含Amp琼脂板───→室温放置,使液体吸收───→37℃倒置培养12~16h。

次日观察转化效率。

图2 质粒结构图谱
图3感受态制备中的热应激
2.2.3质粒DNA提取
随机选择阳性克隆,用LB液体培养基进行扩增,然后按照质粒提取试剂盒操作说明提取质粒,如图4~图8。

(1)取1-5ml细菌培养物,12000rpm离心1min,尽量吸除上清(菌液较多时可以通过多次离心将菌体沉淀收集到一个离心管中)。

(2)向留有菌体沉淀的离心管中加入250μl溶液Ⅰ,使用涡旋振荡器彻底悬浮细菌细胞沉淀。

注意:如果菌块未彻底混匀,会影响裂解导致质粒提取量和纯度偏低。

(3)向离心管中加入250ul溶液Ⅱ,温和地上下翻转6-8次使菌体充分裂解。

注意:混匀一定要温和,以免污染细菌基因组DNA,此时菌液应变得清亮粘稠,作用时间不要超过5 min,以免质粒受到破坏。

(4)向离心管中加入350ul溶液Ⅲ,立即温和地上下翻转6-8次,充分混匀,此时会出现白色絮状沉淀。

12000rpm离心10 min,用移液器小心地将上清转移到另一个干净的离心管中,尽量不要吸出沉淀。

注意:溶液Ⅲ加入后应立即混合,避免产生局部沉淀。

如果上清中还有微小白色沉淀,可再次离心后取上清。

(5)将上一步所得上清液加入吸附柱中(吸附柱加入收集管中),室温放置2分钟,12000rpm离心1min,倒掉收集管中的废液,将吸附柱重新放回收集管中。

(6)向吸附柱中加入700ul漂洗液,12000rpm离心1min,弃废液,将吸附柱放入收集管中。

(7)向吸附柱中加入500ul漂洗液,12000rpm离心1min,弃废液,将吸附柱放入收集管中。

(8)12000rpm离心2min,将吸附柱敞口置于室温或50℃温箱放置数分钟,目的是将吸附柱中残余的漂洗液去除,否则漂洗液中的乙醇会影响后续的实验如酶切、PCR等。

(9)将吸附柱放入一个干净的离心管中,向吸附膜中央悬空滴加50-200ul经65℃水浴预热的无内毒素洗脱液,室温放置5min,12000rpm离心1min。

(10)为了增加质粒的回收效率,可将得到的洗脱液重新加入吸附柱中,室温放置5min,12000rpm离心1min。

质粒提取后置于-20℃保存,并进行凝胶电泳,见图9。

图4 随机挑取单克隆菌落
图5 单克隆扩增后形成的菌液
图6 收集沉淀细菌
图7 加入溶液I后进行涡旋振荡
图8 质粒提取
图9 加样电泳
3结果
3.1 感受态细菌转化效率观察
比较对照平皿和转化平皿,转化平皿中有白色菌落生长,表面光滑,数目较多,
估算菌落数>104 个,表明转化效率较高,见图10、图11和图12。

图10 感受态DH5α涂氨苄琼脂板生长结果
图11 感受态DH5α于未加抗生素琼脂板上生长结果
图12 质粒转化后生长氨苄抗性琼脂板上的DH5α菌落
3.2 质粒提取结果观察
经电泳后,取出凝胶,在凝胶成像仪上观察,DNA 存在处显荧光条带。

如图5所示,1道为DMarker III ,最大条带为4500 bp ,2,3,4,5道表示提取的质粒,条带较亮。

试验只提取4株阳性菌落进行扩增和提取质粒DNA,结果均为阳性。

见图13。

图13 质粒电泳结果图
1 2 3 4 5
4 讨论
4.1 DNA 重组受体系统
目前, DNA 重组技术中应用最广泛的受体系统是大肠杆菌系统。

影响转化效率的因素很多, 其中感受态细胞的转化能力是关键。

大肠杆菌感受态细胞转化方法, 目前主要有氯化钙法、醋酸锂法、电激法等多种手段。

而氯化钙法由于操作简单, 成本较低, 因此在常规实验中应用最为广泛[8]。

用于氯化钙转化法的大肠杆菌菌株主要有JM109, HB101, XL1-blue和DH5α等,国内对于大肠杆菌HB101的感受态制备条件优化鲜有报道。

感受态细胞菌种的来源对转化效率的影响非常显著。

实验结果表明, 对于大肠杆菌HB101, 取自保存于-70℃的原始菌种, 其转化效率最高。

因此, 制备感受态时, 要尽量选取超低温状态下保存的原始菌种, 而经过多次继代的菌种, 由于菌种的衰退, 其转化效率明显下降。

4.2感受态细胞的制备
感受态细胞的制备是分子生物学实验中的一项基本操作, CaCl2法制备过程中需要2次4℃下10 min离心,离心时间较长,且要求实验室具备冷冻离心机。

大肠杆菌感受态建立的过程中,Ca2+是感受态建立和转化的必要条件。

刘志洪[9]研究表明,大肠杆菌在用CaCl2溶液处理后,胞外Ca2+可大量进入胞内,且进入细胞的钙离子量与胞外钙离子浓度相关,对数生长前期的细胞与对数生长后期和稳定期的细胞相比,胞内自身Ca2+浓度低,但其摄取外源Ca2+的能力最强,这应该与其生理代谢活性是相关的,而且这一时期是大肠杆菌细胞最易建立人工诱导感受态的时期。

虽然Ca2+、Mg2+、Sr2 +在元素周期表中处于同一主族,Mn2+与Ca2+处于同一周期,但这些离子诱发产生的感受态,其转化效率都比Ca2+低,而当这些离子与Ca2+按一定比例组合时却只有Ca2+、Mg2+的组合诱导的转化效率升高,其它组合都会使转化效率下降,说明Ca2+具有不可完全替代性。

有人研究[10 - 13]显示Ca2+能与细胞膜形成复合物以利于外源DNA的渗入,在这个过程中金属离子是必需的,PHP也是重新合成然后整合到膜上,因此感受态形成过程可能有内在调节机制而不是完全依靠人工诱导。

42℃热刺激短暂处理细菌细胞时,细胞膜的液晶结构发生剧烈扰动[14],形成间隙有利于质粒DNA分子进入细胞,但若只采用热激处理还是不能形成感受态。

PEG处理细胞,能够影响细胞膜的通透性促进质粒DNA与感受态细胞膜结合[15],适当的溶菌酶也可使细胞壁变薄或形成
孔隙,但单用它们处理大肠杆菌JM109时都不能形成感受态,与CaCl2配合制备感受态,转化效率不升高反而下降,因此,感受态的产生也不是简单的膜结构改变。

4.3 质粒转化效率
转化是将外源DNA分子引入受体细胞,使之获得新的遗传性状的一种手段,它是微生物遗传、分子遗传、基因工程等研究领域的基本实验技术。

转化过程所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体,它可以容忍外源DNA分子进入体内并稳定地遗传给后代。

受体细胞经过一些特殊方法(如电击法、CaCl2 、RbCl 等化学试剂法)的处理后,细胞膜的通透性发生了暂时性的改变,成为能允许外源DNA分子进入的感受态细胞。

进入受体细胞的DNA分子通过复制,表达实现遗传信息的转移,使受体细胞出现新的遗传性状。

将经过转化后的细胞在筛选培养基中培养,即可筛选出转化子。

为了提高转化效率,要考虑以下几个重要因素:(1)细胞生长状态和密度。

不要用经过多次转接或储于4℃的培养菌,最好从-70℃或-20℃甘油保存的菌种中直接转接用于制备感受态细胞的菌液。

细胞生长密度以刚进入对数生长期时为好,可通过监测培养液的OD600 来控制。

DH5α菌株的OD600 为0.5时,细胞密度在5×107 个/ml左右(不同的菌株情况有所不同),这时比较合适。

密度过高或不足均会影响转化效率。

(2)质粒的质量和浓度。

用于转化的质粒DNA应主要是超螺旋态DNA。

转化效率与外源DNA的浓度在一定范围内成正比,但当加入的外源DNA的量过多或体积过大时,转化效率就会降低。

一般情况下,DNA溶液的体积不应超过感受态细胞体积的5%。

(3)试剂的质量。

所用的试剂,如CaCl2 等均需是最高纯度的(GR.或AR.),并用超纯水配制,最好分装保存于干燥的冷暗处。

(4)防止杂菌和杂DNA的污染:整个操作过程均应在无菌条件下进行, 所用器皿, 如离心管, tip头等最好是新的,并经高压灭菌处理,所有的试剂都要灭菌,且注意防止被其它试剂、DNA酶或杂DNA所污染, 否则均会影响转化效率或杂DNA的转入, 为以后的筛选、鉴定带来不必要的麻烦。

参考文献
[1] 萨姆布鲁克J,弗里奇E F,曼尼阿蒂斯T. 分子克隆实验指南[M]. 金冬雁,黎孟枫,译. 2版. 北京:科学出版社,1995.
[2] DOWERW J,MILLER J F,RAGSDALE CW. High efficiency transforma2tion of E. coli by high voltage electroporation [J]. Nucleic Acids Res,1988, 16: 6127 - 6145..
[3] POTTER H. Application of electroporation in recombinantDNA technology[ J ]. Methods Enzymol, 1993, 217: 461 - 483
[4] 周国林,付士红,梁国栋. 同一质粒转入感受态细胞的四种方法转化效率的比较研究[ J ]. 微生物学免疫学进展, 1998, 26 (2) : 20. [5] 姚伟,周会,徐景升,等. 质粒DNA 小量提取法的改进[J]. 应用与环境生物学报,2005(6):122-124.
[5]王立良,王淼,陈连凤,等. 冷冻复苏法高效转化大肠杆菌[J]. 生物技术通讯,2002,13(3):194.
[6] 游雷鸣,翁海波,韩绍印,等. 大肠杆菌DH5α感受态形成因素分析[J]. 生物技术,2007,17(2):37-40
[7] 谢志雄,欧剑虹,赵儒铭,等. Ce3+对大肠杆菌感受态建立和转化的影响[J]. 稀土,2003,24(4):53-56.
[8]张岚岚, 徐春燕, 徐昌杰. 大肠杆菌感受态细胞转化能力的影响因素[ J] . 细胞生物学杂志, 2004, 26( 4 ): 429- 432.
[9] 刘志洪,李文化,沈萍,等. Fura-2荧光探针研究Ca2+对大肠杆菌细胞的跨膜作用[J]. 化学学报,2004,62(4):445-448.
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[13] 萨姆布鲁克,D W拉塞尔. 分子克隆实验指南[M]. 3版,北京:科学出版社,2002:87-96.
[14] 柯涛, 张勇法, 潘园园,等. 温度对大肠杆菌感受态细胞的影响[J]. 河南农业大学学报,2003,37(1):57-60.
[15] Reusch R N,Sadoff H L. Putative structure and functions of a poly - beta-hydroxybutyrate Pcalcium polyphosphate channel in bacterial plasma membranes[J]. Proc Natl Acad Sci USA , 1988 , 85(12):4176-4180.
致谢
首先,在这里我要感谢我的导师李吉霞老师。

可以说,这篇论文如果没有她的悉心指导和严格监督是不能顺利完成的。

李老师平时工作繁忙,但是秉着对学生认真负责的态度,从论题的选定到实验的进行到论文的完成,她都始终坚持和我们在一起,对我们进行监督、指正并且总是耐心解答疑问。

她十分关切我的论文进展情况,总是不断与我交流,在我遇到困难的时候及时指导我去解决。

在我的论文初步完成时,李老师一丝不苟对我论文的每个细节都进行了多次修改,并给我提出了很多有用的意见和建议,让我受益匪浅。

然后我要感谢大学四年来所有教过我的老师,是他们倾囊相授才让我学到了很多知识,并打下了坚实的专业基础。

同时我还要感谢我的同学和我的室友们,感谢他们对我在学习和生活上的支持和帮助。

同时我要感谢我自己,我在做毕业论文中认真学习和积极主动的态度也是我论文能顺利写完的一个很大因素,因此,我也要对自己说,谢谢!
最后我还要感谢母校四年来对我的大力栽培。

谢谢母校奉献我成长的环境,谢谢母校给予我跟随各位老师学习的机会,谢谢母校赠与我与各位同学相识相知的机会!
在此,我要奉上我深深地敬意!谢谢!
附录1:外文文献原文
A home made kit for plasmid DNA
mini-preparation
Simeon Oloni KOTCHONI, Emma Wanjiru GACHOMO, Eriola BETIKU, and
Olusola Olusoji SHONUKAN
Many methods have been used to isolate plasmid DNA, but some of them are time consuming especially when extracting a large number of samples. Here, we developed a rapid protocol for plasmid DNA extraction based on the alkaline lysis method of plasmid preparation (extraction at pH 8.0). Using this new method, a good plasmid preparation can be made in approximately one hour. The plasmids are suitable for any subsequent molecular applications in the laboratory. By applying the recommendations to avoid contaminations and to maximize the plasmid yield and quality during extraction, this protocol could be a valuable reference especially when analyzing a large number of samples.
Key words: Plasmid extraction, PCR, restriction enzymes, sequencing, contamination.
INTRODUCTION
Over the past few years, plasmid DNA has been recognized as the most powerful tool in several biotechnological methods. Based on its molecular properties (closed circular molecule, easily restrictable, harboring an ideal marker gene for selection and rapidly amplifiable in a host system independently), plasmid DNA has been at the center of the most recent and advanced technologies in many disciplines including medicine, agriculture, molecular biology, industry, and biocontrol. In research, plasmid DNA is used as a vector allowing the study and generation of Genetically Modified Organisms (GMO) i.e. the transfer and the subcloning of transgenes across boundaries of species, and functional characterizations of several genes (genomics) within a species. The use of high quality plasmid DNAs often determines the success in various manipulations of genetic material during routine applications such as polymerase chain reaction (PCR) amplification, DNA sequencing, and subcloning of transgenes. Therefore, protocols for extraction of plasmid DNA with high yield and quality have been give serious attention (Sambrook et al., 1989). Several methods for plasmid DNA preparation usually known as plasmid mini-prep, or plasmid DNA miniprep (Birnboim, 1983; Hansen et al., 1995), and commercial kits have been made available. However, some of these methods give relatively low yield and are time consuming especially when carrying out molecular analysis of a large number of samples.
In practice, problems are often linked to the isolation of pure (high quality) plasmid DNA. These problems often arise due to contamination by phenolic compounds and polysaccharides. Acidic polysaccharides are potential inhibitors of Hind III restriction enzyme (Do and Adams, 1991) and they also inhibit classical primer PCR by inhibiting Taq DNA polymerase activity (Demeke and Adams, 1992; Fang et al., 1992;
Pandey et al., 1996). These contaminations distort the results in many analytical applications and therefore lead to wrong interpretations. In our laboratory, we are engaged often in a large number of plasmid preparations for several purposes such as subcloning of transgenes, PCR amplifications, transformation, gene cloning and the screening of positive clones. Therefore, we found it necessary to develop a rapid and efficient protocol for plasmid preparation, enabling us to handle many samples in one experiment, and increasing the number of replicates per day. The protocol described here is relatively simple and rapid. It provides high yield and quality plasmid DNA. It is consistently restrictable, amplifiable by PCR, suitable for cloning and for sequencing. The yield of the plasmid DNA using this protocol is higher than that obtained with commercial kits. a competent E. coli. The following procedure works remarkably well with 2 ml culture of E. coli.
1. Set up a 2 ml overnight culture of E. coli harboring the plasmid in
2.5 ml eppendorf tubes with appropriate antibiotic for the amplification of the plasmid. It is necessary to inoculate this culture with a single colony of transformed E. coli.
2. Harvest the cells by centrifugation at 5000 g for 5 min at room temperature. If one needs to stop the protocol and continue later, the cell pellets can be stored at -20 _C.
3. Resuspend the bacterial pellet in 200 _l of Solution I (see recipe) containing 4 _g/ml lysozyme (from a lysozyme stock of 20 mg/ml in 10 mM Tris-HCl, pH 8.0). Note: Lysozyme must be freshly added to solution I. Moreover, it will not work efficiently if the pH of the solution is less than 8.0. Incubate the suspension at room temperature for 5 min.
4. Add 400 _l of freshly prepared solution II (see recipe) and mix well by inverting gently 4 to 6 times to avoid breaking the plasmid. Do not vortex.
5. Immediately add 200 _l of solution III (see recipe) and mix very gently by pipetting up and down and incubate at 4 _C or in ice for 5 min without shaking. Note: After successfully completing step 5, a white precipitate must form and hang in suspension in the sample, otherwise
no plasmid DNA will be recovered at the end of the procedure. Incubating the mixture in ice enhances the precipitation. The precipitated material contains genomic bacteria DNA, proteins, cell debris and SDS. The solution should be mixed thoroughly to avoid localized ammonium dodecyl sulfate precipitation.
6. Centrifuge at 10000 g for 5 min at room temperature (before loading the samples in the centrifuge, they should be mixed again) and carefully transfer the supernatant into a new eppendorf tubes. Avoid transferring the white debris with supernatant otherwise, step 6 must be repeated.
7. Add 0.6 volume of isopropanol i.e. 0.6 ml isopropanol for 1 ml of supernatant from step 6. Mix gently by inverting 4 to 6 times and keep at room temperature for 10 min.
M pBs pPC87 M pBin
Figure 1. Agarose gel profile of plasmid DNA preparations of different molecular sizes. RESULTS AND DISCUSSION
The method described here presents several advantages. It allows preparation of a large number of samples (24 samples) in a maximum of two and half hours. The time required for the plasmid prep using this method depends on the number of samples to be extracted. Approximately 50 min to one hour is the average time required to complete a good plasmid miniprep with this procedure. It is thus possible to carry out several trials in a day. Generally, the number of the extracted samples in an experiment is strictly determined by the capacity of the centrifuge rotor to be used. Using a centrifuge with 24-eppendorf tubes rotor-holder
M PC 1.3 kb PCR M PC PCR productMP
Figure 2. PCR amplifications of different size of DNA fragments from pBin 19 using
appropriate primers.
we were able to obtain rapidly high quality plasmid preps in our laboratory. As recombinant DNA techniques have advanced, it has become unnecessary, for most purposes, to purify large quantities of plasmid DNA. For example, cleavage with restriction endonucleases, ligation, transformation, and even DNA sequencing can be carried out on relatively small quantity but pure plasmid DNA obtained from small-scale (2 ml) cultures. Using our plasmid miniprep protocol, satisfactory quality was obtained. This indicates that the procedure works well for both small and large size plasmid DNA (Figure 1). The plasmid yield was quantified at 260/280 nm and the concentrations obtained ranged between 2 and 2.8 _g/ _l, which is two to three times more than the concentration obtained using commercial kits. This new method also provides good plasmid for PCR amplification. Arbitrarily, a set of oligonucleotides (primers) was designed for amplification of 460 and 1300 bp DNA fragments using PCR, in order to ascertain the quality of the plasmid prep in subsequence applications. Satisfactory amplification results were obtained (Figure 2). We were also able to sequence our transgenes using MPC1.3 kb PCR product0.4 kb PCR productMPC
the recombinant plasmid preparations (results not shown).
For the precipitation of the plasmid absolute ethanol is often used. In this procedure isopropanol was used (step 7), which also gives a good and high yield of plasmid. It is worthy to note that isopropanol is much cheaper than ethanol. Polysaccharides and other secondary compounds and cell debris released after cell disruption co-precipitate easily with plasmid DNA if the procedure is performed under cold conditions (4 to 8 _C), which finally leads to a viscous plasmid preparation. Such plasmid is usually neither restrictable nor suitable for analytical applications. It often remains in the wells during electrophoretic separation. This protocol works perfectly in the summer and less efficient in cold months especially in winter. It is therefore recommended to use 37 _C wherever room temperature is below 20 _C. The best way to prepare a good plasmid was to carry out the procedure without refrigeration. The use of cold room will lead to a contaminated preparation. We therefore suggest that the protocol would be especially suitable for tropical zones where the temperatures are generally between 23 and 28℃
REFERENCES.
【1】Birnboim HC (1983). A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100:243-255.
【2】Demeke T, Adams RP (1992). The effect of plant polysaccharides and buffer additives of PCR. BioTechniques 12:332-334.
【3】Do N, Adams RP (1991). A simple technique for removing the plant polysaccharide contaminants from DNA. BioTechniques 10:162-166.
【4】Fang G, Hammar S, Grumet R (1992). A quick and inexpensive method for removing polysaccharides from plant genomic DNA. BioTechniques 13:52-54. 【5】Hansen NJV, Kristensen P, Lykke J, Mortensen KK, Clark BFC (1995). A fast, economical and efficient method for DNA purification by use of a homemade beads column. Biochem. Mol. Biol. Intl. 35:461-465.
【6】Pandey RN, Adams RP, Flournoy LE (1996). Inhibition of random amplified polymorphic DNAs (RAPDs) by plant polysaccharides. Plant Mol. Biol. Reptr. 14:17-22.
【7】Sambrook J, Fritsch EF, Maniatis T (1989). Molecular cloning; a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
附录2:外文文献原文译文
质粒DNA试剂盒的研制
许多方法已被用来分离质粒 DNA,但有些是耗费时间,特别是当提取大量的样本。

在这里,我们开发了一种快速的基于碱裂解方法的质粒制备 (ph 值 8.0 提取)的质粒 DNA 提取方案。

使用这种新方法,可以在大约一小时取得好质粒制备效果。

质粒是适合随后在实验室中研究的任何分子应用。

实际中应用,以避免污染,并最大限度地提高质粒的产量和提取过程中的质量,这个协议可能是一个有价值的参考,尤其是当分析大量样品时。

关键词:质粒提取限制性内切酶 PCR 排序污染
前言
过去的几年中,在生物技术的几种方法中质粒DNA 被视为最强大的工具。

基于其分子的属性(闭合环状分子,容易进入独立隐藏的完整标记基因的选择,并在宿主系统迅速扩增独立)。

质粒DNA已经在许多学科,包括医药,农业,分子生物学,工业和生物防治的最新和最先进的技术中心。

在研究中,质粒 DNA 用于允许研究与一代的基因修饰生物体(GMO),即转让和跨边界的物种和物种内部的几个基因 (基因) 的功能特性亚克隆转基因载体。

在日常应用,往往决定使用高质量的质粒DNA中的遗传物质,能使各种操作成功,如聚合酶链反应(PCR)扩增,DNA测序和转基因的亚克隆。

因此,为高产优质质粒DNA的提取协议一直给予严重关注(Sambrook et al., 1989)。

质粒DNA通常被称为质粒小型的准备,或质粒DNA小量(Birnboim, 1983; Hansen et al., 1995)编制的几种方法,商业试剂盒先已提供上市。

然而,这些方法具有相对产量较低的和费时的缺点,尤其是在进行分子分析和大量样本分析。

在实践中,问题往往与高品质质粒DNA有关。

由于酚类化合物和多糖的污染,这些问题经常出现。

酸性多糖对HindⅢ限制性内切酶潜在抑制性(Do and Adams, 1991)和抑制Taq DNA聚合酶活性(Demeke and Adams, 1992; Fang et al., 1992; Pandey et al., 1996)。

在许多分析应用,这些污染物曲解的结果,并因此导致错。

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