正常人体淋巴细胞cDNA文库的构建

应用GIBCOBRL建库试剂盒建立了正常人体淋巴细胞cDNA文库。取新鲜的正常人外周血,分离出淋巴细胞,进行体外培养,提取总RNA,纯化mRNA,并将其反转录成cDNA,与SalI和NotI接头连接后插入λZipLox载体,体外包装后转染到Y1090宿主菌中,进行滴度测试及文库扩增。构建的正常人淋巴细胞cDNA文库含2-6×106重组子,克隆效率为5×1012重组子/g cDNA,插入片段长度约为1~5kb。扩增后的文库浓度为3×107重组子/μl,将文库稀释到10-6时所产生的噬菌斑密度最为适宜。试验结果表明,该库符合标准,所构建的正常人淋巴细胞cDNA文库为进一步筛选目的基因、制作基因芯片等提供了有效的工具。 Abstract:A lymphocyte cDNA library of normal human was constructed in order to obtain specific gene and prepare lymphocyte gene chips to detect the relative genes between psychiatric diseases and immunity.The lymphocyte was abstracted from fresh normal human blood and cultured in vitro.Total RNA of lymphocyte was extracted from the cultured cells and then mRNA was extracted further.Moreover,single-strand cDNA and double-strand cDNA were synthesized in turn.The double-strand cDNAs were ligated to SalI and NotI adaptor,which were later ligated to arms of λZipLox.Ligated-cDNAs were packed in vitro,and then infected E.coli Y1090.Titering the phage and amplifying the library.The lymphocyte cDNA library consisted of 2-6×106 recombinants with the length of 1~5kb and the cloning efficiency was 5×1012 recombinants/g cDNA.The amplified library was 3×107recombinants/μl in concentration and the number of bacteriophage plagues was the most suitable in density after it was diluted to 10-6 in concentration.The constructed cDNA library of normal human lymphocyte would be helpful to further detecting target genes and preparing gene chips etc.     

用限制性cDNA文库制作K562细胞基因表达谱芯片探针

以人红白血病K562细胞为材料,应用限制性显示PCR（RD-PCR）技术构建cDNA文库,该文库通过PCR引物3′端延伸两个不同碱基形成136对引物对cDNA进行限制性扩增,得到136组不同的PCR扩增产物,纯化后与载体连接并转化细菌,即为限制性cDNA文库,根据不同的分组进行克隆的鉴定和分离。并进行大量扩增制备cDNA芯片探针,该方法构建的文库因经过了限制性分组扩增,每组均含有特定的cDNA,因而大大加快了随后克隆的分离 和鉴定的速度,为基因芯片探针制备提供了一个新方法。     

发现新基因的高效方法——cDNA文库的复性式均一化技术

由复性式均一化技术制作的均一化cDNA文库（equalized cDNA library,normalized cDNA library）是近年来发展起来的一种获得EST、发现新基因的高效平台。本文就该技术的原理、方法比较、存在问题和展望进行了阐述。 Abstract:The cDNA library normalized by reassociation is a newly-developed,effective platform for EST acquisition and gene discovery.This papper presents the principle,procedure,comparison,deficiencies,application and future of the technique of the normalization.     

一种改进的cDNA文库固相构建法

本文介绍一种新的cDNA文库固相构建法。文库构建过程中,cDNA的合成和修饰全部在固相介质――磁珠上完成,简便、迅速、文库质量高,能满足不同目的的cDNA文库构建,是一种值得借鉴和应用的好方法。 Abstract：A method for cDNA library construction was introduced.All enzymatic steps of library synthesis were performed on a solid support-magnetic beads.Therefore it combines fast speed and convenient with high quality library,making it ideally suited for most purposes.It is a good method and worth learning and utilization.     

快速简便筛选cDNA文库的SSS法

建立了一种快速简便筛选cDNA文库的方法—SSS法（subsection screening）。该方法用cDNA噬菌体平板划块分组和根据目的基因设计的一对特异性引物,用PCR技术逐级筛选cDNA文库获得目的基因。与其它筛选cDNA文库的方法相比,该方法范围可控,目标明确,易于获得目的基因,而且快速、简捷、省时,一般可在一周内筛选出目的基因。本实验室利用该方法在半个月内筛选出了一个查耳酮异构酶(chalcone isomerase)(CHI)基因,一个黄酮类化合物3′-羟化酶(flavonoid 3′hydroxylase)(F3′H)基因,一个热激蛋白(heat shock protein)(HSP)基因和一个1 484 bp 热激蛋白基因片段。此方法用于同时筛选多个基因,可收到事半功倍的效果,对其它文库筛选也有借鉴意义。 Abstract:A quick and simple method subsection screening (SSS) method for screening cDNA library by PCR was established.With this method,cDNA phage plate was cut into several blocks and a couple of primers was designed according to target gene.And then the target genes were obtained by screening cDNA library.Comparing with other methods,this method has many advantages such as controlled range and clear target,and also quick and simple for obtaining the target genes.It is possible to get a target gene in one week in general by this method.Thus,the CHI gene,F3′H gene,HSP gene and one HSP partial fragment,were obtained respectively in half month in our lab.We can get twice the result with half the effort when we screen several genes in the same time.It is also suitable to screen other libraries.     

家蝇cDNA文库的构建

自Ｂｏｍａｎ小组 (Ｓｔｅｉｎｅｒ ,1981)从惜古比天蚕(Ｈｙａｌｏｐｈｏｒａｃｅｃｒｏｐｉａ)中分离、纯化出第一种抗菌肽天蚕素以来 ,人们已在昆虫和其他无脊椎动物中发现了 170多种抗菌肽 (Ｌｏｗｅｎｂｅｒｇｅｒｅｔａｌ ,1999)。目前 ,人们不仅搞清楚了多数抗菌肽的氨基酸序列、结构和功能特点 ,而且还对一些抗菌肽的基因进行了克隆 (陈留存和王金星 ,1999)。我们以家蝇为材料 ,分离、纯化出了抗菌肽 ,在此基础上 ,构建了经过诱导的家蝇ｃＤＮＡ文库 ,以克隆其基因。1　材料和方法1 1　实验材料家蝇 (Ｍｕｓｃａｄｏｍｅｓｔｉｃａ)…     

香猪肌肉组织cDNA文库的构建及其EST测序成功率的分析

以香猪背最长肌为实验材料,以Lambda ZAP II为载体,构建了肌肉组织的cDNA文库。结果表明,cDNA文库的滴度在3.4×107 pfu/ml以上,重组率在94%以上,插入片段大小平均在1.5kb以上。同时指出,如果从3′端测序,多于30 个碱基T的插入片段是造成ESTs测序成功率低的主要因素。 Abstract:Using Longissimus Dorsi muscle as material and Lambda ZAP II as Vector,Xiang Pig Longissimus Dorsi muscle cDNA library has been constructed in our study.The results showed that the tritation of the library was 3.4×107 pfu/ml,the recombinant percentage was 94%,and the fragment length of inserted average cDNA were 1.5kb.The study pointed out that the more than 30 T insertion is the major factor for low percetage if sequencing the 3′-end.     

枸杞果实cDNA文库的构建

目的 :通过构建枸杞果实cDNA文库 ,分离类胡萝卜素合成酶基因。方法 :枸杞果实RNA的提取采用改进的异硫氰酸胍 -酚 -氯仿法 ,利用PolyATractmRNAIsolationSystem(Promega公司 )分离mRNA ,然后利用UniversalRibocloneRcDNASynthesisSys tem(Promega公司 )合成双链cDNA ,在cDNA末端连接上EcoRⅠ接头 ,并与λExCell载体连接 ,利用PackageneLambdaDNAPackagingSystem进行包装。结果 :首次构建出滴度为 8 4× 10 4 pfu ml,重组效率为 86 9%的枸杞果实cDNA文库。结论 :构建出枸杞果实cDNA文库 ,为类胡萝卜素合成酶基因的分离奠定了基础     

cDNA文库固相构建法

本文介绍一种cDNA文库的固相构建法。文库构建过程cDNA的合成和修饰全部在固相介质—磁珠上完成,简便、迅速、文库质量高,能满足不同目的的cDNA文库构建,是一种值得借鉴和应用的好方法。     

cDNA文库的构建策略及其应用

cDNA文库在基因分离和克隆中具有重要的作用。从cDNA文库中能筛选出所需要的目的基因,并直接用于该目的基因的表达。cDNA文库是发现新基因和研究基因功能的基础工具。随着分子生物学技术的发展。cDNA文库构建方法有了很大改进和提高,就cDNA文库的构建方法及其应用进行综述。     

一种改进的构建杂交扣留cDNA文库的方法

介绍了一种改进的利用减法杂交构建λ噬菌体cDNA文库的方法,该方法利用处理的sscDNA减去两种对照的mRNA富集目的sscDNA并用磁珠法分离杂交单体,结果表明其增加了杂交单体的产量、简化了实验步骤,而且更有利于富集所需基因,是构建cDNA文库的一个有效改进.     

菠萝果实cDNA文库的构建

以菠萝幼果为研究材料,采用Creator^TMSMART^TM cDNA Library Construction Kit技术构建菠萝幼果cDNA文库,其文库的库容量为1．01×10^6,重组率为92％,PCR检测表明大多数插入片段均大于1000bp。