2,332
edits
Griffin:shRNA Transfection: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
→Concentration and purity of nucleic acids
| Line 213: | Line 213: | ||
Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids. | Determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids. | ||
'''DNA:'''In terms of plasmid preparation, [http://mcmanuslab.ucsf.edu/lentivirus_RNAi.html McManus Lab] has not observed a need to use E. coli cells that are highly defective for recombination | '''DNA:'''In terms of plasmid preparation, [http://mcmanuslab.ucsf.edu/lentivirus_RNAi.html McManus Lab] has not observed a need to use E. coli cells that are highly defective for recombination. High DNA quality usually means high transfection efficiency. All DNA preparations should be performed by Cesium prep or endotoxin-free ion exchange plasmid purification methods. If poor transfection is consistently observed, it may be worth performing a additional clean-up of the DNA. The transfection protocols described here are sensitive to the amount of DNA. It is important to optimize DNA:Transfection Reagent ratios. | ||
===Transfection in serum-free media=== | ===Transfection in serum-free media=== | ||
