限制性酶切与连接

重组质粒的构建重组质粒的构建是基因工程的核心步骤之一，其目的是将目的基因插入到质粒载体中，以实现目的基因的稳定表达和克隆化。

以下是重组质粒构建的主要步骤：1.目的基因获取首先需要获取目的基因。

目的基因可以从基因文库、PCR、基因组测序等方法中获取。

根据需要选择合适的方法，将目的基因克隆到质粒载体中。

2.载体质粒选择选择适合的质粒载体是重组质粒构建的关键步骤之一。

根据目的基因的特点和表达要求，选择适合的质粒载体。

常见的质粒载体有pET、pUC、pBluescript等。

3.限制性酶切限制性酶切是重组质粒构建的重要步骤之一。

通过限制性酶切，将目的基因和质粒载体分别切开，露出粘性末端，以便于连接反应。

4.连接反应将切好的目的基因和质粒载体的粘性末端连接在一起，形成重组质粒。

连接反应需要使用T4DNA连接酶或其它连接酶进行催化。

连接反应需要在适宜的温度和pH 条件下进行一定时间，以确保重组质粒的正确构建。

5.转化宿主细胞将连接反应得到的重组质粒转化到宿主细胞中。

常见的宿主细胞有细菌、酵母、昆虫等。

转化方法有多种，如电穿孔法、化学转化法等。

转化后需要在适宜的培养条件下进行培养，以获得大量的重组质粒。

6.克隆筛选克隆筛选是重组质粒构建的重要步骤之一。

通过克隆筛选，可以确定重组质粒是否正确构建。

常见的克隆筛选方法有蓝白斑筛选、酶切法等。

7.序列验证最后需要对重组质粒进行序列验证，以确保目的基因的正确插入和序列的准确性。

序列验证可以通过Sanger测序等方法进行。

实验三、酶切与连接一、实验目的与原理简介限制性内切酶在基因工程中主要应用地以下两个方面：制作基因酶切图谱和进行基因克隆。

制作基因图谱，就是利用特定的酶切出特定的条带；而利用基因克隆时选择酶应注意以下几个方面：1）克隆片段的长度；2）克隆片段中切点的情况3）载体上切点的情况；4）切割与连接方式；5）接头状态。

酶切方式可分为部分酶切和完全酶切两种：1）部分酶是指同一DNA 片段上有些被切开而另一些未被切开，此法主要应用于基因 的克隆 。

用部分酶切法是基于基因内部可能有此酶的位点。

进行部分酶切可通过两个方式：一是不同的时间内在同一酶反应管中取样终止反应，利用时间来控制酶切的程度。

另一种是在其余条件相同时控制 酶的稀释度，利用不同酶浓度控制酶切程度，这种方法因易于控制反应而被广泛应用。

2）完全酶切法适用于如载体切割、酶切图谱的制作、基因的鉴定与DNA 片段的分离工作。

完全酶切又可分为单酶切、多酶切两种。

在多酶切反应中当2种或2种以上的酶有相同 的反应条件时，可同时进行酶切，不然须在前一种酶作用完成后将其失活，而后进行第二种酶切反应，这样可以避免片段混乱现象的出现。

二、材料和试剂限制性内切酶NotI 、EcoRI ；10×Buffer ， PCR 产物、pPIC9K 质粒、10×T4连接Buffer 、T4 Ligase 、DDW 、琼脂糖、电泳缓冲液 、Goldview 染液、胶回收试剂盒；电泳仪、恒温水浴锅、EP 管、移液枪、灭菌枪头、紫外检测仪三、实验步骤1）PCR 产物双酶切（NotI ，EcoRI ），pPI9K 质粒双酶切(NotI ，EcoRI )；PCR 体系如下：2）然后电泳检测后在紫外检测仪下观察（UV ，260nm ）。

3）切胶回收（尽量不要切到不含目的片段的胶），按照胶回收试剂盒标准操作。

4）回收产物电泳检测后进行连接：连接体系： 10×T4连接Buffer 1μl目的基因 6μl质粒载体 2μl产物酶切体系pPI9K 质粒酶切体系 DDW4.6μl DDW 7μl 10×HBuffer1μl 10×H Buffer 1μl 目的片段4μl pPI9K 质粒 1.6μl NotI quickcut 0.2μl NotI quickcut 0.2μlEcoRI quickcut 0.2μl(37℃15min) EcoRI quickcut0.2μl(37℃15min)T4 Ligase 1μl混合后16℃，过夜连接。

实验四限制性内切核酸酶的酶切与鉴定一、实验原理限制性内切酶是一类能识别双链DNA分子中特异核苷酸序列的DNA水解酶，主要存在于原核生物中。

根据限制酶的识别切割特性、催化条件及是否具有修饰酶活性可分为Ⅰ、Ⅱ、Ⅲ型三大类。

其中Ⅱ类酶在分子克隆和基因操作中最为有用，是常用的分子生物学工具酶。

限制性内切酶识别序列长度一般为4～8个呈回文序列的特异核苷酸对。

一般情况下，识别序列越长，在同一DNA分子中识别位点出现的频率就越小。

许多限制性内切酶的酶切位点已被确定。

例如EcoRl 酶的识别与切割序列为以下6个碱基对。

5′……GAATTC……3′3′……CTT AAG…… 5′这些末端为互补的，即粘性末端，并可在连接酶的催化下与由EcoR I产生的其它分子末端相连接。

限制性内切酶主要用于基因组DNA的片段化、重组DNA分子的构建与鉴定、载体中目的基因片段的分离与回收以及DNA分子物理图谱的构建等。

根据酶切目的和要求不同，可有单酶切、双酶切或部分酶切等不同方式。

根据酶切反应的体积不同，可分为小量酶切反应和大量的酶切反应。

小量酶切反应主要应用于质粒的酶切鉴定，体积为20 μl, 含0.2～1 μg DNA，大量酶切反应用于制备目的基因片段，体积为50～100 μl，DNA用量在10～30ug。

本实验为EcoR I对质粒pUC18的小量酶切。

在质粒的双链环状DNA分子上有多个限制性内切核酸酶酶切位点。

在用特定的限制性内切核酸酶对质粒进行酶切反应后，通常可采用琼脂糖凝胶电泳进行鉴定酶切效果。

二、仪器与试剂1.仪器：水浴锅、离心管、移液器、吸头、电泳设备等。

2.试剂：质粒pUC18、EcoR I限制性内切核酸酶、内切酶反应缓冲液、琼脂糖、电泳缓冲液、6×上样缓冲液、溴化乙啶染液、无菌水等。

限制酶的多种切割方法 一、错位切割含目的基因的DNA片段和运载体──形成黏性末端 1. 单酶切 （1）同一种限制酶分别切割含目的基因DNA片段与运载体。

这是最简单的一种方法，含目的基因的DNA片段与运载体在同种限制酶作用下形成相同的黏性末端，这两个黏性末端间能通过碱基间的配对而连接，进而形成重组DNA分子。

（2）用同裂酶去切割含目的基因DNA片段与运载体。

有一些来源不同的限制酶识别核苷酸顺序和切割位置都相同，这类酶称为同裂酶（同切酶或异源同工酶）。

同裂酶产生同样的切割，形成相同的黏性末端。

2009年江苏生物高考第34题就出现了同裂酶（BamHⅠ与BstⅠ），其中的差别只在于当识别顺序中有甲基化的核苷酸时，一种限制性内切酶可以切割，另一种则不能。

（3）用同尾酶切割含目的基因DNA片段与运载体。

有一些来源不同、识别的核苷酸顺序和切割位置各不相同，但能产生相同的黏性末端，这类酶被称为同尾酶。

常用的限制酶BamHⅠ、BglⅡ、BclⅠ和Sau3A就是一组同尾酶，它们切割DNA之后都形成由GATC四个核苷酸组成的黏性末端。

显而易见，由同尾酶所产生的DNA片段，是能够通过其黏性末端之间的碱基互补作用而彼此连接起来。

【例1】（2005年天津理综）：限制性内切酶Ⅰ的识别序列和切点是—G↓GATCC—，限制性内切酶Ⅱ的识别序列和切点是—↓GATC—。

在质粒上有酶Ⅰ的一个切点，在目的基因的两侧各有1个酶Ⅱ的切点。

①请画出质粒被限制酶Ⅰ切割后所形成的黏性末端。

②请画出目的基因两侧被限制酶Ⅱ切割后所形成的黏性末端。

③在DNA连接酶的作用下，上述两种不同限制酶切割后形成的黏性末端能否连接？为什么？ 解析：这两种酶属于同尾酶，能产生相同的黏性末端，因此能够连接起来。

③由于两者产生的黏性末端相同（都是GATC），黏性末端之间能发生碱基互补配对而连接。

（4）单酶切的优点与缺点。

单酶切优点就是操作简单。

它的缺点主要有两个：①酶切后的质粒与目的基因在DNA连接酶作用下易自身环化：用单酶切切割后的目的基因和质粒的两侧各有一个黏性末端，而且是相同的末端，因此这两个末端会发生碱基互补配对而使目的基因和质粒分别发生首尾相连，都形成环状DNA分子，其中质粒又恢复成没有切割前的样子。

酶切连接法
酶切连接法是一种分子生物学技术，用于将不同的 DNA 片段连接在一起。

该方法通常包括以下步骤：
1. 酶切：使用特定的限制酶将待连接的 DNA 片段进行切割，产生特定的粘性末端。

2. 纯化：通过凝胶电泳或其他方法，将切割后的 DNA 片段进行纯化，去除杂质和未切割的 DNA。

3. 连接：将纯化后的 DNA 片段与载体 DNA（如质粒或病毒载体）在连接酶的作用下进行连接。

连接酶能够将粘性末端连接在一起，形成新的 DNA 分子。

4. 转化：将连接后的 DNA 分子导入宿主细胞中，如大肠杆菌或酵母细胞。

转化可以通过热激法、电穿孔法或化学转化法等方法进行。

5. 筛选：通过选择性培养基或其他方法，筛选出含有连接后的 DNA 分子的宿主细胞。

6. 鉴定：对筛选出的宿主细胞进行鉴定，确认其中含有连接后的 DNA 分子。

酶切连接法是一种常用的 DNA 克隆技术，可以用于构建基因文库、表达载体、基因敲除等实验。

该方法具有高效、简便、快速等优点，是分子生物学研究中不可或缺的技术之一。

DNA的酶切与连接1. 实验目的了解DNA限制性内切酶和连接的作用原理，学习和掌握利用限制性内切酶进行DNA 消化和片段的方法和技术。

2. 实验原理DNA限制性内切酶和连接酶是遗传工程中实现DNA的切割和重组的重要工具酶，也是基因工程技术赖以生存的基础。

1970年Smith H·O等从流感病毒中提取了第一个限制性内切酶Hind II，其作用于外源DNA后，切割产生平末端。

人们经过研究发现，限制性内切酶可以识别双链DNA分子上的特异序列，通常识别区具有回纹结构，并使两个特定核苷酸之间的磷酸二酯键断裂。

目前已经从微生物中发现了200多种限制性内切酶，其中大部分可以切割DNA形成粘性末端。

如EcoR I，可以识别6个碱基的序列：▼5’ ――――――GA TTC――――――3’3’ ――――――CTTAAG――――――5’▲切割后形成的片段5’――――――G AA TTC――――――3’3’――――――CTTAA G――――――５’当限制性内切酶作用于DNA时可以形成的酶切片段数为：片段数目= 切点数+1 （线状DNA）片段数目= 切点数（环状DNA）切点出现的频率为1/4s（S为识别顺序所含的碱基数目）DNA连接酶则可以将切开的DNA片段连接起来，此时需要接口两端具有磷酸根。

对粘性末端的单链可以进行点接，对于平末端来说也可以进行连接，但是需要较多的酶。

在基因工程中，可以利用同一种限制性内切酶分别切割目标DNA和运载体，然后利用T4DNA 连接酶将目标序列整合到运载体中，使DNA中的3’-OH与5’-P生成磷酸二酯键。

3. 实验用具及材料电泳仪、恒温水浴锅、紫外检测仪、微量移液器、Eppendorf离心管10×酶切反应缓冲液、T4DNA连接酶缓冲液、溴酚蓝指示液、EB电泳缓冲液、PBR322质粒DNA、pXZ6质粒DNA，λphage DNA、DNA Marker EcoRI Hind III。

质粒DNA酶切、连接、转化、筛选、鉴定(2011-04-29 10:42:22)转载▼质粒DNA酶切、连接、转化、筛选、鉴定实验目的1、学习和掌握限制性内切酶的特性2、掌握对重组质粒进行限制性内切酶酶切的原理和方法3、掌握利用CaCl2制备感受态细胞的方法4、学习和掌握热击法转化E.coli的原理和方法5、掌握α互补筛选法的原理6、学习用试剂盒提取重组质粒DNA的方法7、复习琼脂糖凝胶电泳的原理及方法实验原理重组质粒的构建需要对DNA分子进行切割，并连接到合适的载体上进行体外重组。

限制性核酸内切酶和DNA连接酶的发现与应用，为重组质粒的构建提供了有力的工具。

限制性核酸内切酶酶切分离法适于从简单基因组中分离目的基因。

质粒和病毒等DNA 分子小的只有几千碱基，大的也不超过几十万碱基，编码的基因较少，获得目的基因的方法也比较简单。

DNA连接酶催化两双链DNA片段相邻的5’-磷酸和3’-羟基间形成磷酸二酯键。

在分子克隆中最有用的DNA连接酶是来自Ｔ４噬菌体的DNA 连接酶：T4 DNA连接酶。

T4 DNA连接酶在分子克隆中主要用于：1、连接具有同源互补粘性末端的ＤＮＡ片段；2、连接双链DNA分子间的平端；3、在双链平端的DNA分子上添加合成的人工接头或适配子。

目的DNA片段与载体DNA片段之间的连接方式（以T4DNA连接酶为例）主要有以下几种：（一）、具互补粘性末端片段之间的连接大多数的核酸内切限制酶都能够根据识别位点切割DNA分子，形成1~4核苷酸单链的粘性末端。

当载体和外源DNA用同一种限制性内切酶切割时，产生相同的粘性末端，连接后仍保留原限制性内切酶的识别序列；如果用两种能够产生相同的粘性末端的限制酶（同尾酶）切割时，虽然可以有效地进行连接，但是获得的重组DNA分子消失了原来用于切割的那两种限制性核酸内切酶的识别序列，这样不利于从重组子上完整地将插入片段重新切割下来。

（二）、平末端的连接载体分子和外源DNA插入片段并不一定总能产生出互补的粘性末端。

ＤＮＡ的限制性酶切反应实验目的学习在实现DNA的体外重组过程中，正确选择合适的载体和限制性核酸内切酶，并利用限制性核酸内切酶对载体和目的DNA进行切割，产生利于连接的合适末端。

通过对DNA的酶切，学会设计构建重组DNA分子的基本方法，掌握载体和外源目的DNA酶切的操作技术。

实验原理核酸限制性内切酶是一类能识别双链ＤＮＡ中特定碱基顺序的核酸水解酶，这些酶都是从原核生物中发现，它们的功能犹似高等动物的免疫系统，用于抗击外来ＤＮＡ的侵袭。

限制性内切酶以内切方式水解核酸链中的磷酸二酯键，产生的ＤＮＡ片段５’端为Ｐ，３’端为ＯＨ。

根据限制酶的识别切割特性，催化条件及是否具有修饰酶活性可分为Ⅰ、Ⅱ、Ⅲ型三大类。

Ⅰ类和Ⅲ类限制性内切酶，在同一蛋白分子中兼有甲基化作用及依赖ATP的限制性内切酶活性。

Ⅰ类限制性内切酶结合于特定识别位点，且没有特定的切割位点，酶对其识别位点进行随机切割，很难形成稳定的特异性切割末端。

Ⅲ类限制性内切酶在识别位点上切割，然后从底物上解离下来。

故Ⅰ类和Ⅲ类酶在基因工程中基本不用。

Ⅱ型酶就是通常指的ＤＮＡ限制性内切酶。

它们能识别双链ＤＮＡ的特异顺序，并在这个顺序内进行切割，产生特异的ＤＮＡ片段；Ⅱ型酶分子量较小，仅需Mg2+作为催化反应的辅助因子，识别顺序一般为４～６个碱基对的反转重复顺序；Ⅱ型内切酶切割双链ＤＮＡ产生３种不同的切口－－５’端突出；３’端突出和平末端。

体外构建重组DNA分子，首先要了解目的基因的酶切图谱，选用的限制性核酸内切酶都不能在目的基因内部有专一的识别位点，即当用一种或者两种限制性核酸内切酶切割外源供体DNA时，能够得到完整的目的基因。

其次，选择具有相应的单一酶切位点的质粒、噬菌体等载体分子作为克隆的载体。

常用的酶切方法有双酶切法和单酶切法两种。

本实验采用单酶切法，即只用一种限制性内切酶切割目的DNA片段，酶切后的片段两端将产生相同的黏性末端或平末端，再选用同样的限制性内切酶处理载体。

酶切酶连原理
酶切酶连原理是一种常用的基因克隆技术，通过利用限制性核酸内切酶和DNA连接酶，将目的基因与载体DNA进行切割和连接，从而构建重组的DNA分子。

首先，使用限制性核酸内切酶对目的基因和载体DNA进行酶切，产生具有特定粘性末端的DNA片段。

这些片段具有相同的粘性末端，可以通过DNA连接酶将其连接在一起。

在连接过程中，需要确保目的基因与载体DNA的粘性末端序列相互匹配，以确保正确的重组。

连接后的重组DNA分子可以转入宿主细胞中进行表达或保存。

此外，为了提高酶切酶连的效率和特异性，通常会使用特定的缓冲液和交联剂。

同时，为了去除未酶切和未连接的DNA片段，需要进行电泳和胶回收等操作。

总之，酶切酶连原理是基因工程中常用的技术之一，可用于构建基因表达载体、基因敲除载体、基因治疗载体等，具有重要的应用价值。

限制性内切酶酶切的常见问题及解决方法限制性内切酶酶切的常见问题及解决方法，个人觉得总结得很好，特转贴供本供大家分享。

酶切现问题，看内切酶说明书，相应试剂公司目录。

不同公司出产的内切酶，菌株来源、制备工艺、纯度活力、酶切活性优化可能不同，。

可在上面找到酶单位定义、保存条件、酶切体系[buffer及与其它酶双切的buffer等]、酶切反应温度[有些酶是在50、55或30等温度下反应的]、酶是否受甲基化影响、是否有星号活性及出现星号活可能因素、保护性碱基，同尾酶、同裂酶1. 酶切不开或不完全1.1 质粒问题纯度差或残留酶切抑制物最为常见。

杂蛋白存在会影响酶切，表现为A260/A280低于1.8；抑制物常见酚、盐、乙醇等；【重新提DNA，使用可靠试剂盒或可靠手工提取试剂，1.2 酶的问题：确认内切酶有效 [很多内切酶虽然有过期时间，但过期后只要能够有效酶切，可用。

确认酶切效果不好，做标记，更换] 。

【题外话：用内切酶注意】a. 内切酶如无特殊要求，保存-20~-30度。

并非越低越好，酶通常是保存在50％甘油缓冲液中，温度过低时，酶会发生冻结（运输过程例外，由于酶需要低温运输，所以方便的情况下是使用干冰，酶会冻结，但冻结次数有限，相对是一种比较好的选择），如果是经常使用的话，酶会被反复冻融，从而降低了活性。

当然如果温度过高，呵呵，你就自己想去吧。

b. 酶在使用时应置于冰盒中取酶。

这点大家都清楚，不过多强调也没坏处。

因为实验室有些同学，酶取出后是放在冰盒上的，但有两点忽略了：拿酶的时候，手不是抓着管子上端，而握在管子的底部，相当于用手在给酶进行加热；有些酶是放在冰盒中，但吸酶的时候，还是将酶拿出来。

偶尔一次没有大碍，反复如此，可能会影响酶活。

c. 酶使用完后应尽快旋紧盖子。

有时我们可以发现，即使是-20~-30度放置，酶仍然会冻结。

个人认为的可能原因是由于在配置酶切反应时，酶管的盖子在较长时间的开着，甘油会吸取空气中的水分，对于常用的大包装的酶有时就会出现冻结现象。

限制性核酸酶的名词解释限制性核酸酶（Restriction Endonucleases）是一类在细菌和一些原核生物中存在的酶，它们能够特异性地识别DNA的特定序列并切割DNA链。

它们在基因工程和分子生物学研究中发挥着重要作用。

限制性核酸酶的发现和研究始于20世纪50年代，由福尔塞特（Werner Arber）和史密斯（Hamilton Smith）等科学家共同开创。

他们发现，引起细菌某些基因位点变异的机制不仅仅涉及基因突变，还存在着一种特殊的酶系统。

这种系统能够识别和切割细菌中的外源DNA，从而限制其他生物的基因物质进入宿主细胞。

限制性核酸酶的命名来源于其作用机制的特征。

它们能够特异性地识别DNA中的一段短序列（通常为4-8个碱基对），这个识别序列被称为限制性切割位点或识别位点。

当限制性核酸酶识别到与其特异性结合的DNA序列时，会产生两种反应：剪切反应和保护反应。

剪切反应是指限制性核酸酶在切割位点两侧的磷酸二酯连接处切割DNA链，形成两个断片。

保护反应是指限制性核酸酶通过与自身产生的一种酶抑制蛋白结合，在宿主细胞中保护其自身DNA不被切割。

限制性核酸酶的特异性结合和切割机制是其应用于分子生物学研究和基因工程的基础。

通过将限制性核酸酶作用于DNA分子，可得到特定的DNA片段。

这些DNA片段可以进一步用于DNA测序、PCR扩增、基因克隆和DNA分析等实验操作。

此外，限制性核酸酶还广泛应用于DNA指纹图谱的建立和遗传变异的分析等研究领域。

限制性核酸酶的分型标准是根据它们的识别序列和切割位点特征来分类的。

目前已经发现了数千种限制性核酸酶，其中一部分具有特殊的识别和切割特性，成为了分子生物学实验中常用的酶。

例如，EcoRI是最早被发现的一种限制性核酸酶，它能够识别5'-G↓AATTC-3'序列，并在A和T之间的碱基之间切割。

此外，还有BamHI、HindIII、XhoI等常用限制性核酸酶，它们具有不同的识别序列和切割位点。

2009年初三英语质量检测试卷（满分150分，考试时间100分钟）考生注意：本卷有9大题，共106小题。

试题均采用连续编号，所有答案务必按照规定在答题纸上完成，做在试卷上不给分。

Part 1 Listening (第一部分听力)I. Listen and choose the right picture (根据你听到的内容，选出相应的图片): (共6分)A B C DE F G1.________2. _______3. ________4. ________5. ________6. ________II. Listen to the dialogue and choose the best answer to the question you hear (根据你听到的对话和问题，选出最恰当的答案): (共10分)7.A) By bus. B) By underground.C) By car. D) By bike.8. A) They’re going to see a film. B) They’re going to stay at home.C) They’re going to have a walk in the park. D) They’re going to watch TV.9. A) She is a secretary. B) She is a teacher.C) She is a writer. D) She is an engineer.10. A) The traffic was terrible. B) The telephonedidn’t work..C) The man was too late. D) The man didn’t know the time.11. A) In hospital. B) At home.C) In the library.D) At the bookstore.12. A) 3yuan.B) 5yuan.C) 12yuan.D) 10 yuan.13. A) March 14.B) March 13.C) March 12. D) March 11.14. A) Rainy. B) Sunny. C) Warm. D) Cold.15. A) Once a month. B) Once a year.C) Twice a month. D) Twice a year.16. A) Taxi driver and passenger. B) Conductor and passenger.C) Husband and wife. D) Customer and salesman.III. Listen to the passage and tell whether the following statements are true or false (判断下列句子是否符合你听到的内容, 符合的用“T”表示，不符合的用“F”表示): (共7分)17. The recent Australiaforest fires killed many people and animals.18. Akoala was saved by a fireman, and he has bee a star.19. Sallywas the name of the badly burnt koala.20. A fireman made a video of the koala with his mobile phone.21. A lot of people got to know the koala from the Internet.22. It was difficult to get so close to a koala.23. The koala will go back into the wild in about five weeks.IV. Listen to the dialogue and fill in the blanks (根据所听到的对话完成下列句子，每空格限填一词):（共7分）24. Alice lived at Flat 701, Block 2, ______Garden Estate.25.Alice would like to order a pizza with some ______.26.Alice’s telephone number was _______.27. Alicesaid to the boy that she wanted a ______ pizza.28. Alice asked for two ______ of apple juice and three ice creams.29. The boy told Alice that the total ______ was 132 yuan30. The boy would ______ the food to her house in half an hour.Part 2 Vocabulary and Grammar（第二部分词汇和语法）V. Choose the best answer (选择最恰当的答案，用A、B、C或D表示): (共26分)31. Kitty is such ______ honest student that she is often praised by her teachers.A) a B) an C) the D) /32. I’ve got two tickets ______ tonight’s show. Would you like to go with me?A) of B) on C) with D) for33. Bret, an old friend of ______, has decided to give up drinking because he is a driver now.A) my father B) my fathers C) my father’s D) my fathers’34. He had money, so he asked for free rides from many people on the way.A) much B) many C) few D) little35. About _______ of the clerks in this office are under the age of 40.A) three-fifths B) thirdfifths C) third-five D) threefive36. Although their class teacher is very strict ______ them, they still love her very much.A) in B) at C) with D) to37. ______ of his parents works in Shanghai, so he lives with his grandparents.A) No one B) Both C) None D) Neither38. Billy didn’t get to the airport before 3:00 in the afternoon, ______?A) did heB) does he C) didn’t he D) doesn’t he39. A survey showed that Jingjing and Huanhuan were ________ among the five Olympicmascots.A）popular B）more popular C）most popular D）the most popular40. Take your glasses with you, ______ you can’t enjoy the performance.A) orB) so C) and D) for41. I’m very sorry to hear that you failed the exam. The underlined part means ______ .A) passed B) missed C) didn’t pass D) didn’t miss42. Joe left for Los Angeles ______ he got his uncle’s telephone call.A) now that B) as soon as C) although D) so that43. Why does he look so _________? Did he sleep well last night?A) interested B) frightened C) tired D) surprised44. “Would you like me to show you the way?” the volunteer said ______ to the tourist.A) lovely B) likelyC) kindly D) friendly45. The managerlost his temper because his assistant made the same mistake again. Theunderlined part means ______ .A) got angry B) became happy C) went wrong D) was surprised46. Tom bought a new MP4 last week. It ______ him nearly 200 yuan.A) paidB) spent C) took D) cost47. Our monit or can’t go to the cinema because she _____ a foreigner around our school.A) shows B) was showing C) is showing D) has showed48. Shanghai Wild Animal Park ______ many visitors every year.A) attracted B) attracts C) has attracted D) will attract49. Look at that sign. You______ walk on the green grass.A) mustn’t B) needn’t C) canD) may50. The newspaper says that more money ______ fighting pollution next year.A) will spend B) will be spent C) would spend D) would be spent51. The gate-keeper told Jackson _____ out of school between classes.A) don’t go B) didn’t go C) not go D) not to go52. Though it looked like rain this morning, it has ______ to be a fine day.A) turned up B) turned over C) turned on D) turned out53.—______ will the astronaut be away from the earth?—For about one month.A) How soon B) How long C) How often D) How much54. The old man couldn’t remember .A) where he puts hiscupB) where had he put hiscupC) where he had put hiscup D) where did he put hiscup55.—Happy birthday, Kitty. Here’s a present for you.—______A) Thank you. The same to you. B) Thank you. It’s lovely.C) It’s a pleasure. D) You’re wele.56. —You’d better not eat too m any sweets, John. It’s bad for your teeth.—______A) Thanks. I’ll takeyour advice. B) That’s all right.C) Of course not.D) It doesn’t matter.VI. plete the sentences with the given words in their proper forms( 用括号中所给单词的适当形式完成下列句子)：(共8分)57. We call people from Germany ______. (German)58. Please help ______ to the apples on the table, Mary. (you)59. When John was in his ______ , he became a very popular writer. (forty)60. ______ , we were not caught in the heavy rain last night. (lucky)61. I hear that Mr Brown is too ______ to remember his own age. (forget)62. The scientists have just discovered a new kind of ______. (ill)63. Miss Black hopes that she will ______ in finding a good job soon. (success)64. We all know that it is ______ for an ordinary plane to reach the moon. (possible)VII. Rewrite the sentences as required（按要求改写句子，每空格限填一词）：（共12分）65. The new car costs Miss Liu much money. (改为否定句)The new car ______ ______ Miss Liu muchmoney.66. John took part in the English contest last month. (对划线部分提问)______ ______ John take part in last month?67. It’s very convenient to travel by undergroun d between Pudong and Puxi. （改为感叹句）______ ______ it is to travel by underground between Pudong and Puxi!68.Mr Zhang is not only our teacher but also our friend. （保持原句意思基本不变）Mr Zhang is our teacher and friend ______ ______.69. You should write all the answers on the answer sheet. (改成被动语态)All the answers should ______ ______ on the answer sheet.70. Little Tom asked his mother, “Does the sun rise in the east?” （保持原句意思基本不变）Little Tom asked his mother ______ the sun ______ in the east.Part 3 Reading and Writing (第三部分读写)VIII. R eading prehension (阅读理解): (共56分)A. True or False（判断下列句子是否符合短文内容，符合的用“T”表示，不符合的用“F”表示): ( 7分)Gary was a policeman. He liked boiled eggs. He often had one for breakfast. One morning in December, he was on duty on the road in his police car. At ten o’clock, he went into a small caféby the roadside for breakfast. He asked for a boiled egg, a drink and a sandwich. When the waiter brought them to him, he found that the egg was not cooked enough. He decided to cook his egg a little longer. He put the egg inside the microwave oven and turned the switch on. He let the eggcook for one minute. Then he took it out and went back to his table. He picked up his spoon and pushed it into the egg.Suddenly there was a loud explosion (爆炸声). For a moment, all the other people rushed to hide under the tables in the caféas they thought someone had thrown a bomb (炸弹) into the building. But when they saw Gary’s face, they all laughed because he had egg all over it. Gary had made a big mistake as eggs must never be cooked in a microwave oven.71. Gary often had a boiled egg for breakfast.72. One morning, he had breakfast in a small café by the roadside.73. A waitress brought a boiled egg, a drink and a sandwich to him.74. He cooked the egg inside the microwave oven because it was not cooked enough.75. All the other people rushed out of the café when they heard a loud explosion.76. People were frightened because someone had thrown a bomb into the building.77. Gary’s face looked funny because it was all covered with egg.B. Choose the best answer（根据短文内容，选择最恰当的答案): ( 7分)A four-year-old girl is lying in the arms of a doctor. She has just bee motherless. A bomb destroyed her home and killed her mother.Every day we see pictures like this on TV or in newspapers. We see young lraqi children begging for food and water from American and British soldiers. They move through towns and cities towards the capital, Baghdad. We see these children following grown-ups, carrying bags of things almost the same size as their small bodies as they run away from their homes.“Dad, why are the Americans attacking us? Are we going to be killed?” asks the son of Abu Sinar, an Iraqi engineer. Abu finds it hard to explain this war to his eight-year-old son.He tries to fort him by saying, “The bombs are far away from us. The Americans are fighting the soldiers. We are going to be all right.” Abu knows this isn’t always the truth, but what else can he do? In Baghdad, parents give their children sleeping pills to make them sleep so that they won’t hear the sound of bombing. And all the schools are closed.“All we can do is to listen to and hear the war,”said Abu. “People in Baghdad, men and women, young and old, have to live in fear. We don’t know where the next bombs are going. We can do nothing but wait to be killed.”78. The four-year-old girl’s mother was killed by ______.A. some Iraqi soldiersB. a gunC. a man in the hospitalD. a bomb79. What pictures can we see on TV or in newspapers?A. Iraqi soldiers and people are fighting the Americans.B. Iraqi children are begging for food and water.C. American and British soldiers are attacking Iraqi children.D. American and British soldiers are carrying bags of things as they run.80. The children have to run away from their homes because ______.A. their parents tell them to do soB. they want to help the wounded soldiers.C. their homes are not safe to stay inD. they want to fight the American soldiers81. Abu Sinar ______.A. is an American engineerB. knows he and his family will be killed in the warC. finds it difficult to answer his son’s questions about the warD. doesn’t know how to fort his son82. In Baghdad, parents give their children sleeping pills so that they will ______.A. not hear the sound of bombingB. not feel hungry any longerC. stay safe in their homesD. go to school the next day83. During the war, children in Baghdad are ______.A. still studying at schoolB. living a terrible lifeC. are fighting at the frontD. safer than their parents84. From the passage, we know that people in Baghdad are ______.A. helplessB. braveC. unhealthyD. confidentC.Choose the words or expressions and plete the passage（选择最恰当的单词或词语完成短文):（14分）Each nation has many volunteers who help to take care of others. They read books to the people in hospitals or homes for the aged. Sometimes they just visit them and play games with them or listen to their ___85___.Other young volunteers go and work in the homes of people who are sick or old. They paint, clean up, or ___86___ their houses, do their shopping or mow their lawns. For boys who no longer have ___87___ there is an organization called Big Brothers. College students and other men takethese boys to baseball games or fishing places and help them to get to know things that boys ___88___ learn from their fathers.Each city has a number of clubs where boys and girls can go to play games or learn crafts (工艺,手艺). Some of these clubs show movies or organize ___89___ to the mountains, beaches, museums or other places of interest nearby. Most of these clubs use a lot of high school and college students as volunteers because they are young enough to ___90___ the names of boys and girls.Volunteers believe that the ___91___ people in the world are those who help to bring happiness to others.85. A. songs B. speeches C. voices D. problems86. A. build B. repair C. make D. plan87. A. brothers B. sisters C. fathers D. mothers88. A. almost B. usually C. hardly D. never89. A. short trips B. long journeys C. expensive visits D. exciting flights90. A. report B. choose C. select D. remember91. A. luckiest B. cleverest C. happiest D. busiestD.Read the passage and fill in the blanks with proper words（在短文的空格内填入适当的词，使其内容通顺，每空格限填一词，首字母已给):（14分）There are many online food shopping sites. Things l___92___ CDs, books, and DVDs sell well online. But it is quite different to sell food online b___93___ people like to see food with their own eyes. It is very i___94___ for people to buy fresh fruit and vegetables. Customers like to c___95___ their food by its color. They also want to touch and smell it. Some people like going to the same shop to buy food with good quality. So if the food is always g___96___ , they will feel fortable buying it online.Boxes of fruits and vegetables have been delivered to h___97___ around Britain for a few years now. Dried or canned foods are almost the same all the time, but fresh things are not. For example, people may want to pick their own apples so they can take the o__98__ that look nice. Buying dried and canned food online is easier. A person can buy this kind of food on the Internet and then buy fruits and vegetables at a store or a market.E. Answer the questions（根据短文内容回答下列问题): ( 14分)Christmas is an important festival in the West. People decorate their houses with lights andChristmas trees. They also hang mistletoe in their homes. It is a custom that when a man and a woman find themselves under the mistletoe, they must kiss! Mistletoe is a symbol of forgiveness and love.Decorations for Chinese New Year are mainly red and gold. Red is thought to be lucky. Red lanterns and firecrackers are popular and childrenare dressed in new red clothes. Decorated peach trees are also mon and poems are used to decorate doorways to bring luck in the New Year.At Halloween, decorations are mostly frightening! Ghosts, witches and monsters are used to decorate just about everything. Jack-o’-lanterns are another important decoration for Halloween. They are carved out of pumpkins (南瓜) and a candle is lit inside.At some festivals, animals are decorated. For example, in Austria, the Almabtrieb festival happened each year in autumn. The cows are led down from the mountains to stables (牛棚) for the winter and they are decorated to celebrate. In Thailand and India elephants are often decorated for festivals and weddings.99. At Christmas, what must a man and a woman do if they meet under the mistletoe?100. Why are red lanterns and firecrackers popular at Chinese New Year?101. What decoration can you find in the doorways at Chinese New Year?102. Which word from Paragraph 3 can we use to describe ghosts, witches and monsters?103. What are Jack-o’-lanterns made of?104. It isn’t mon to find a decorated elephant at a wedding in Thailand, is it?105. How many festivals have been talked about in the passage?Ⅸ. Writing (作文): (共18分)106. Write a passage of at least 60 words on the topic “A successful person in my eyes ”. ( 以“我眼中的成功人士”为题写一篇不少于60个词的短文，标点符号不占格。

一、限制性内切酶酶切注意事项在进行限制性酶切反应过程中，影响限制性酶切反应效果的因素较多，要设计酶切反应，一般均应考虑诸如酶切系统、温度条件、反应体积、底物性质及星型反应等因素。

根据各种不同的条件，酶切反应的设计一般应注意以下问题：1. 大多数限制酶贮存在50%甘油溶液中，以避免在-20 C条件下结冰。

当最终反应液中甘油浓度大于 12％时，某些限制酶的识别特异性降低，从而产生星活性，更高浓度的甘油会抑制酶活性。

因此加入反应的酶体积不超过反应总体积的1/10，避免限制酶活性受到甘油的影响。

2. 浓缩的限制酶可在使用前用1邓艮制酶缓冲液稀释，但切勿用水稀释以免酶失活，用水稀释的酶不能长期保存。

3. 反应体系中Mg2+是限制酶仅有的共同因子，当用其它二价阳离子代替Mg2+或加入能螯合Mg2+的EGTA或EDTA时，酶活性会被抑制，或改变酶的特异性，导致星型反应。

4. 多种因素可引发星型反应：①非最适的pH;②Co2+、Mn2+、2n2+取代 Mg2+;③酶浓度大于25u/ug ；④盐浓度降低；⑤高浓度甘油的存在（＞12%）；⑥有机溶剂的存在。

5. 反应混合物中 DNA底物的浓度不宜太大，小体积中过高浓度的DNA会形成粘性DNA溶液抑制酶的扩散，并降低酶活性。

建议酶切反应的DNA浓度为0.1-0.4ug/ul 。

6. 酶切反应所加入的酶量应适中，根据底物的种类、量的多少和体积的大小而定，对不同的限制酶，各厂家均有一最大的消化量指标可参考。

7. 当要用两种或两种以上限制酶切割DNA时，如果这些酶可以在同种缓冲液中作用良好，则两种酶可同时切割，如果这些酶所要求的缓冲液有所不同，则可采用以下两种替代方法：①先用在低离子强度的缓冲液中活性高的酶切割DNA然后加入适量 NaCl及第二种酶，继续反应;②使用能够使多数内切酶均表现较高活性的单种缓冲液。

8. 用同一种酶切割不同的DNA时，所需酶量不同，可根据DNA底物上酶切位点的多少与ZDNA 存在位点的数目比较后，决定用酶量。