Tools
Procedures
Donor cell
Surface chemistry of host cell-Host cell surface charges either will attract or repell DNA as a result of opposite or similar charges
Presence of cell wall (in the case of bacteria, fungus and plant) causes additional physical barrier to the up-take and entry of DNA
Charges on DNA- negative charge on DNA modulates interaction with the host cell especially cell surface
List of selected agent as potential to make cell competent
Bacterial Strains
Bacterial Strains Streptococcus pneumoniae In B subtilis
Helicobacter pylori
Legionella
Peumophila
E. Coli
Competent agents
Mitomycin C, fluoroquinolone
UV light
Ciprofloxacin
Mitomycin C, norfloxacin, ofloxacin, nalidixic acid, bicyclomycin, hydroxyurea, UV light
Calcium chloride, Rubidium Chloride
Preparation of competent cell Bacteria is incubated with divalent cation (calcium chloride, manganese chloride or rubidium chloride) for 30mins at 4°C
During this process, cell wall of treated bacteria is swell and it gather factors required for intake of DNA docked on the plasma membrane
Fragile
Storage
Centrifugation Speed
Pippeting
Cloning Vector Mechanism of transcription, replication, genomic and cDNA library
Expression Vector Mechanism of translation, protein production - protein
Protein production is a multistep process and an over-view of these steps are as follows