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Coating Microprojectiles with DNA
1.6 m gold particles are prepared using the guidelines for coating microprojectiles
provided in the Bio-Rad instruction manual, except for two modifications.
First, in the agglomeration step, we vortex the gold for a longer period
of time to reduce clumping( 4 minutes for each wash instead of 2 minutes
as recommended). Second, after all of the reagents are added to the gold
in the DNA precipitation step, we vortex for 2 minutes, then intermittently
for the next 20 minutes. The additional time in this step allows for improved
coating of the gold particles.
Coating Macrocarrier Disks with Gold
Transfection of dissociated cells requires only a few simple modifications
to the standard Biolistic bombardment protocol. Because the percentage
of successfully transfected cells varies with the density of gold particles
as well as with cell density, we typically use 1.5 times the standard
microprojectile recipe to coat six macrocarrier disks (i.e. 3 mg gold,
5 g plasmid/macrocarrier). At greater than two or three times the standard
density, however, cell death becomes significant. For co-transfections
of two different expression constructs, one-half of the standard amount
of each DNA construct is used, such that the total amount of DNA used
per recipe remains the same.
Transfecting Cell Lines
To transfect dissociated cells, the media is removed from the culture
dish as thoroughly as possible without completely drying the cells; aspirating
media into a sterile serological pipette works well. Blasting should proceed
quickly from this point, to prevent dessication of the cells. The aspirate
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