蓝白筛选 Identifying transformants foreign DNA insert Ii. Distinguishing cells carrying Insert. conta ining recombinant molecules from cells carrying vectors without inserts Intact vector, voctor no insert with insert origin of tsZ lacZ acz one split by L Transformation: foreign DNA enters the host cell lacZ oone foreign DNA insert. nect n2 pNoev91 Lacz oone transcript a Bl X l selecting cells that have received a plas
蓝白筛选
噬菌体及黏粒载体 Phage and cosmid vectors PhagλDNA Plasmid Incorporate into virus capsules cos sites amp OB Restriction ite Origin of replication Intect E coll host cells Cleave DNA and plasmid with restriction enzyme. Splice cos DNA into plasmid cos sitos Select for ampicillin resistance by Cosmid vector growing infected cells on medium Linearize cosmid at restriction site containing ampicillin Combine with genomic DNA and ligate Genomic DNA strands Surviving cells contain recombinant plasmids which may be cloned Incorporate into virus capsules
噬菌体及黏粒载体
YAC裁体 YAC vectors Plasmid with YAC vector elements Expose yeast ceas to recombinant YACs: a small percentage of YAC entor yeast cells Foreign DNA uses .e 2 awat h eccell Cut with several restriction eery to release two vector arme 389 te with phosphatase. Plate onto medium containing two antibiotics Cells containing YACs with two electable markers survive
YAC载体
How many clones for one human ? genome Vector vg Insert Size of Clones plasmid 1,000bp 3000,000 pl hage 15,000bp 200,000 cosmid 50,000bp 60.000 YAC 200,000bp 15000
How many clones for one human genome? Vector Avg. Insert Size # of Clones plasmid 1,000 bp 3,000,000 phage 15,000 bp 200,000 cosmid 50,000 bp 60,000 YAC 200,000 bp 15,000
cDNA文库 ◆mRNA分离、纯化与分离 ◆cDNA的合成 ◆cDNA末端的处理 ◆与载体的连接
cDNA文库 mRNA分离、纯化与分离 cDNA的合成 cDNA末端的处理 与载体的连接