simple subcloning experiment.transformants are screened most easily by digesting the DNA from mnprpaaionsohernsf6mansfol6adbynasonngroe时 Growth and storage of ransformants Single froma transformation plate are grown in liqui medium,maintaining the antibiotic selection for the plasmid,and a portion of the culture is stored for later use as a frozen glycerol stock. 9.Gel analysis:Recombinant plasmids can be distinguished from vectors by size on an agarose gel and by ned using an agarose gel by digestion of the plasmid with a restriction enzyme known to cut asymmetrically within the insert sequence. Lesson 5 DESIGN OF PLASMID VECTORS (4) (1)教学目的和要求： 题求掌握外题片段与质拉载体的连接及篮白斑篇洗的原理，表达载体及基因的表达。 (2) 教学重点和难点 篮白斑筛选的原理 Ligation products:One of the most important steps in a cloning procedure is to distinguish between recreated vector molecules and recombinant plasmids.A number of methods have been developed to facilitate this resistance:A vector with two antibiotic resistance genes can be used to screen fo recombinants if the target fragment is inserted into one of the genes,thus insertionally inactivating it. Blue-white screening:Insertional inactivation of the LacZ'gene on a plasmid can be used to screen for recombinants on a plate containing IPTG and X-gal The X-gal is converted to a blue product if the Lacz gene is intact and induced by IPTG;hence recombinants grow as white colonies. for cloning. Transcription of cloned inserts:A promoter within the vector may be used either in vivo or in vitro to transeribe an inserted fragment.Some vectors have two specific promoters to allow transeription of either strand ofthe insert Expression vectors:Many vectors have been developed which allow genes within insert to be expressed by transcription from a strong promoter in the vector.In some cases.for example using T expression vectors,a large proportion of the total protein in the E.coli cells may consist of the desired product. Lesson6 BACtERIOPHAGE VECTORS (4) (1)教学目的和要求： 要求掌握噬菌体载体的类型及筛选重组DNA的原理， (2)教学重点和难点： 噬菌体载体的类型及筛选重组DNA的原理。 1.Bacteriophage:The infection and subsquent lysis of E.coi by bacteriophage k may be used to propaga loned DNA fra nts.Non ssential portions of the linear 48 kb genome may be replaced by up to 23 kbof foreign DNA 2.A Replacement vectors:Target DNA fragments are ligated with the A DNA ends,which provide the essential genes for infection,to produce recombinant phage DNAs. 3.Packaging and infection:A packaging extract.consisting of A coat proteins and processing enzymes 4 simple subcloning experiment, transformants are screened most easily by digesting the DNA from minipreparations of the transformants, followed by analysis on an agarose gel. 8. Growth and storage of transformants: Single colonies from a transformation plate are grown in liquid medium, maintaining the antibiotic selection for the plasmid, and a portion of the culture is stored for later use as a frozen glycerol stock. 9. Gel analysis: Recombinant plasmids can be distinguished from vectors by size on an agarose gel and by excising the inserted fragment with the same restriction enzyme(s) used to insert it. Fragment orientation: The orientation of the insert in the vector may be determined using an agarose gel by digestion of the plasmid with a restriction enzyme known to cut asymmetrically within the insert sequence. Lesson 5 DESIGN OF PLASMID VECTORS（4） （1）教学目的和要求： 要求掌握外源片段与质粒载体的连接及篮白斑筛选的原理，表达载体及基因的表达。 （2）教学重点和难点： 篮白斑筛选的原理。 Ligation products: One of the most important steps in a cloning procedure is to distinguish between recreated vector molecules and recombinant plasmids. A number of methods have been developed to facilitate this process. Twin antibiotic resistance: A vector with two antibiotic resistance genes can be used to screen for recombinants if the target fragment is inserted into one of the genes, thus insertionally inactivating it. Blue-white screening: Insertional inactivation of the LacZ' gene on a plasmid can be used to screen for recombinants on a plate containing IPTG and X-gal. The X-gal is converted to a blue product if the LacZ' gene is intact and induced by IPTG; hence recombinants grow as white colonies. Multiple cloning sites: A multiple cloning site provides flexibility in choice of restriction enzyme or enzymes for cloning. Transcription of cloned inserts: A promoter within the vector may be used either in vivo or in vitro to transcribe an inserted fragment. Some vectors have two specific promoters to allow transcription of either strand of the insert. Expression vectors: Many vectors have been developed which allow genes within a cloned insert to be expressed by transcription from a strong promoter in the vector. In some cases, for example using T7 expression vectors, a large proportion of the total protein in the E. coli cells may consist of the desired product. Lesson6 BACTERIOPHAGE VECTORS（4） （1）教学目的和要求： 要求掌握噬菌体载体的类型及筛选重组 DNA 的原理。 （2）教学重点和难点： 噬菌体载体的类型及筛选重组 DNA 的原理。 1. Bacteriophage λ：The infection and subsequent lysis of E. coli by bacteriophage k may be used to propagate cloned DNA fragments. Nonessential portions of the linear 48.5 kb λ genome may be replaced by up to 23 kb of foreign DNA. 2. λ Replacement vectors: Target DNA fragments are ligated with the λ DNA ends, which provide the essential genes for infection, to produce recombinant phage DNAs. 3. Packaging and infection: A packaging extract, consisting of λ coat proteins and processing enzymes