分子生物学9-cloningvectors1.pptVIP

Molecular Biology Basic procedures of subcloning DNA purification Analysis of transformants Alkaline phosphatase (AP) Questions Vectors Cloning vector: ori, MCS, screening marker Expression vector: ori, MCS, screening marker, promoter, SD sequence, terminater Shuttle vector: two ori pBR322 Twin antibiotic resistance for screening of recombinants Insertional inactivation of one of the two antibiotics resistance genes on the plasmid will cause the recombinant lose the resistance to the antibiotics. Replica plating (影印平板) pUC18/pUC19 Expression vector What should be taken into consideration during sub-cloning How to choose restriction enzymes? 1. Analyze the restriction sites in the sequence of the insert gene. 2. Design the primers which carry the restriction enzyme recognition sites. How to isolate transformants from wild type bacteria? antibiotics (amp, tet, neo, kana, cam, zeocin, etc.) How to distinguish clones containing recombinant plasmid from those with self-ligated plasmids？ How to express a recombinant fusion protein ？ Genomic DNA of λ phage λ phage used as a vector Cloning in Charon 4 (1) Cosmid T-DNA introduced to plant cells by Ti plasmid Review questions Why do we need to attach DNAs to vectors to clone them？ Describe the process of cloning a DNA fragment into the BamHI and PstI sites of the vector pUC18. How would you screen for clones that contain an insert? What kind of vector would you use to insert a transgene into a plant such as tobacco? Diagram the process you would use. You want to make a genomic library with DNA fragments averaging about 40 kb in length. Which vector would be most appropriate to use? Why? Describe the use of a baculovirus system for expressing a cloned gene. What advantages over a prokaryotic expression system does the baculovirus system offer? Partial regestriction digestion * 12kb-20kb 75%-105% It is required for the packaging that insert DNA should be between 12~20 kb. Mainly used for construct