Then incubate the tubes at 37 degrees Celsius for at least one hour. Vortex the samples for five seconds and briefly spin them to collect the liquid to the bottom of the tubes. Add 10 microliters of each reagent to each sample tube. To treat the samples with Serratia marcescens endonuclease, prepare mix A and mix B reagents. Then transfer 200 microliters of the supernatant from each tube into labeled micro-centrifuge tubes with screw caps for the dot blot assay. Pellet the cell debris by centrifugation at 3, 700 times G and four degrees Celsius for 10 minutes. Then, vortex the tubes vigorously for one minute to maximize the recovery of AAV from the cells. To recover the viral particles, quickly thaw the frozen tubes in a 37 degree Celsius water bath. Store the samples at minus 80 degree Celsius until use. Then, transfer the suspension into 15 milliliter polypropylene conical tubes. On day five, pipette the cells and virus-containing medium up and down or use a cell scraper to scrape all the cells. On days one and two, under an inverted fluorescence microscope, observe cells transfected with the PCMVGFP plasmid to assess the transfection efficiency. Healthy, low-passage number cells should be used and care should be taken not to disrupt the cells'monolayer. Transfection of HEK 293 cell is a critical component of this protocol for high-titer virus production. Then gently agitate the plates and return them to a 37 degree Celsius incubator with 5%CO2. Following the incubation, briefly spin the tubes in a micro centrifuge to collect the liquid to the bottom.Īnd add the DNA PEI mixture drop-wise to the culture medium in each well of the HEK 293 culture plate. Vortex the tubes briefly and incubate the DNA PEI mixture at room temperature for 15 minutes. The final volume is approximately 100 microliters or 5%volume of the culture medium. Add four microliters of PEI solution to the PBS plasmid DNA mix. As indicated in this table, the total amount of DNA is two micrograms per well. On day zero, after culturing HEK 293 cells to 90%confluency, prepare the transfection reagent by mixing the plasmids in 96 microliters of PBS without calcium or magnesium in sterile 1.5 milliliter micro-centrifuge tubes. Though this method has commonly been used for AAV vector quantitation, it can also be applied to address various fundamental questions about AAV biology as shown in this protocol. The main advantage of this technique is that it is a straightforward inexpensive method that avoids the drawbacks of other AAV titration methods. This method can determine both purified and the non-purified AV vac titers and assess the ability for the AAV AAP to promote assembly of cognate and heterologous AV serotype-capsis.
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