transfection Flashcards
what plasmid are we using in the lab
pCMV DsRed-Express2
what does pCMV DsRed-Express2 encode
encodes the red fluorescent protein from Discosoma sp
what is the pCMV DsRed-Express2 plasmid used for/what characteristics does it have
has a prokaryotic origin and a Janamycin resistant marker. It also has a eukaryotic origin and promoter
describe the method of the lab
co-transfection of the plasmid containing the gene of interest with a plasmid vector (DsRed). after incubation we will look for fluorescence in the cells, which indicates a successful transfection of the plasmid
describe what it means for plasmid DNA needing to be of ‘transfection quality’
cannot be contaminated with genomic DNA or RNA, free from chemicals/salts/phenol, free from contamination by LPS
formula for transfection efficiency
(#cells fluorescing in field of view/total cell # in field of view) x 100
difference between stable and transient transfection
transient: no integration into the host genome, short term, used on a small and analytical scale (ie promoter studies, functional studies, pathway analysis)
stable: integrated into the genome using a selection marker, this is long term, used for medium to large scale protein production for drug discovery, gene therapy, functional studies. It requires selection
4 ways nucleic acids can be introduced into cells
physical methods: electroporation, direct microinjection
viral-mediated transfection: mainly adenovirus, lentivirus, retovirus-based
particle-based: via nanoparticles (ie gold)
chemical methods: calcium phosphate, liposome-mediated (phospholipid or cholesterol bilayer), or use the multicomponent method that uses cationic polymers and lipids
list some parameters that influence transfection efficiency
purity, amount, dilution medium, ratio (uL to ug), type of reagent, timing, amount of complex, type of cell, density and quality, passage number
