Our team of volunteers is shooting rice embryos with the gene gun on Saturdays at Counter Culture Labs.
Sreenivas and Nathaniel from Johns Hopkins University have joined us for the Summer. They provided the first version of the plasmid containing the griffithsin construct which we have been cloning on Fridays. The work on Saturday is to get a layer of plasmid to adhere by forming an adsorption ionic bond with 0.7 micron Au microparticles. This procedure has an element of timing, including vortexing and centrifuging, while keeping everything cool. Here Anthony prepares a centrifuge. The rotor is chilled in a freezer before centrifuging.
First the Au microparticles are washed multiple times with cold
ethanol. The last wash is done with a pH
9.0 borate buffer.
Nathaniel has the pipettes ready with tips and lined up, he will quickly add plasmid, CaCl2, and protamine and vortexed immediately.
This kept cold while allowing to rest for 10
minutes. During this time precipitation
occurs.
After centrifuging the supernatant is removed and the
precipitate is re-suspended in pure ethanol.
Then the particles are carefully pipetted into the cartridges which will be loaded into the gene gun. Here David instructs Sreenivas on the cartridge loading.
The ethanol needs
time to evaporate and then the cartridges can be loaded into the gene gun.
Here’s a closer look at the cartridges after loading.
Rice embryos are placed between 2 wire screens in the nozzle
of the gene gun.
Sreenivas opens the valve to allow the helium gas to fill
the measuring tube of the gene gun before firing.
David furiously takes notes on all of this.
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