南京大学-杨荣武分子生物学6.pptVIP

* * * * * * * * * * * * * * * * * * Phage P1 vectors: cloning up to 100 kb DNA fragments PACs: like P1 vectors but the DNA is not packaged (transfer by electroporation) BACs: Bacterial Artificial Chromosomes Based on the F factor of E. coli: --100 kb plasmid, propagates through conjugation --low copy number (1-2 copies per cell) --2 genes (parA and parB): accurate partitioning during cell division BACs: just have par genes, replication ori, cloning sites, selectable marker Can propagate very large pieces of DNA: up to 300 kb Relatively easy to manipulate: move into cells by transformation (electroporation) General BAC vector replication selection Cloning, etc 7 kb o---- Cloning strategies ----o Making DNA “libraries” (from genomic DNA, mRNA “transcriptome”) Screening to identify a specific clone (the needle in the haystack) -- by the sequence of the clone -- by the structure or function of the expressed product of the clone Course reading: #28 (and 29) Overview of strategies for cloning genes 1) 2) 3) 4) Get DNA Ligate to vector Transform or transfect Look for the gene… Genomic DNA RNA 1) Get DNA Ligate to vector: how to make this reaction favorable? This yields a “library”, a representative set of all the pieces of DNA that make up a genome (or all the cDNAs that correspond to the “transcriptome”) cDNAs from different tissues reflect the different RNA populations that you find in distinct cell types: Hence “liver” vs. “brain” vs. “heart” cDNA libraries There are lots of ways to identify a particular gene… Overview of strategies for cloning genes * * * * * * * * * * * * * * * * * * * * * * Setting up a transformation--how will the competent cells be treated? No plasmid (negative control, nothing should grow on this plate) Supercoiled plasmid of a known concentration (to determine efficiency of competent cells, in transformants/microgram) Vector DNA (dephosphorylated?) ligated without insert DNA (background transformants) Vector DNA ligated