《分子生物学》2-section ggen manipulation-1.pptVIP

Identify the protein product of an interested gene Protein activity Western blotting using a specific antibody In vivo expression and functional assay back Analysis of a clone Restriction mapping: digestion of the with restriction enzymes. Sequencing the cloned DNA back You may have to fully understand the function and application of all the enzymes listed in Table 1 if you want to manipulate genes Enzymes commonly used in DNA cloning Alkaline phosphotase Reverse transcriptase, DNA ligase (T4) DNA pol I (Klenow fragment), T4, Taq Exunuclease III Mung bean nuclease and S1 nuclease Polynucleotide kinase Restriction enzymes: e.g. EcoRI, HindIII RNase A, RNase H T7, T3and SP6 RNA polymerases Terminal transferase back G2. Preparation of plasmid DNA 1. Plasmid as vectors Plasmid minipreparation Alkaline lysis Phenol extraction Ethanol precipitation Cesium chloride gradient purification Plasmid as vectors Plasmids: small, extrachromosomal circular molecules, from 2 to ~200 kb in size, which exist in multiple copies within the host cells. contain an origin of replication and replicate independently Usually carry a few genes, one of which may confer resistance to antibacterial substance. Example: ampr gene encoding the enzyme b-lactamse which degrades penicillin antibiotics such as ampicillin. Plasmid minipreparation from E. coli Plasmids ~2-20 kb in length that is much smaller than E. coli chromosomal DNA (4600 kb), and independently supercoiled Resistant to shearing force and chemical denaturation, thus can be isolated from the chromosomal DNA easily such as by alkaline lysis. Minipreparation (miniprep) Isolation of plasmid DNA from a few mililiters (ml) of bacterial culture. Minipreps Growth of the cells containing plasmids Collect the cells by centrifugation Alkaline lysis resuspension? alkaline lysis ? neutralization Phenol extraction to get rid of the protein contaminants Ethanol precipitation to concentrate the nucleic acids remained (0.3M NaAc, 2-3 vol ethan