DEVELOPMENT AND SCREENING OF HIGH PRODUCING 13 GALACTOSIDASE ACTIVITY BY CLONES OF BHK-21 CELL LINEP. ELLATAH, G. JAYA PRAKASH, GOVARDHAN
Plasmid DNA (pNeoi3Gal) was transformed into DH5a cells (competent cells). The plasmid DNA was extracted by alkaline lysis method. The extracted plasmid DNA was further confirmed by restriction pattern on agarose gel electrophoresis. Purity and quantity of plasmid DNA was determined spectrophotometrically by extracting the DNA using Qiagen tip method. The plasmid DNA was transfected with BHK Cells by calcium phosphate transfection method. These cells were incubated for 48 hours at 37°C in CO2 incubator (5% CO2). After incubation neomycin antibiotic was added to the medium to develop antibiotic resistant clones. These antibiotic resistant clones were cotransfected with FLP recombinase vector (P0G44). The precisely recombined clones exhibited intense blue colour with X-gal staining solution. The i3 Galactosidase activity expressed by these clones, were measured by cell lysis method.
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