# Loading Vials and Setting Initial Conditions

1. For the next few steps, it is recommended to spray your gloves with 70% ethanol.
2. Partially insert autoclaved vials into the eVOLVER Smart Sleeves such that efflux straw is entirely visible, if possible.
3. Start by attaching input lines (clear) to the short influx straw, avoid touching the tips of the lines to avoid contaminating the inside.
4. Next, attach efflux lines (blue) to the long efflux straw. It is critical to check for loose connections or incorrectly routed lines. Failure to do so will cause overflows and potentially damage the Smart Sleeve.
5. In the setup menu in the electron app, run all pumps in 10 second intervals to fill the vials with media. Run enough media so that the efflux pumps start removing media through the efflux straws.
6. Visually inspect the culture volumes at this point. If the efflux straws do not appear to be functioning efficiently (media levels rise above the efflux straw), inspect the fluidic line connections and the peristaltic pump. Correct or replace parts as needed to prevent overflows.
7. Push vials down until fully encased by the Smart Sleeve. If aluminum sleeves are cut to different lengths, vials may appear to be covered in different amounts.
8. Be sure that the desired calibration files have been selected from the dropdown menu in the setup panel. These files are stored on the device itself, and will default to the most recently used calibration. Temperature and OD calibrations must be repeated if vials are replaced, and OD calibrations are recommended if switching to a new organism (i.e. bacteria vs yeast). Otherwise, calibrations can be used over and over again.
9. Set the initial conditions for experimental parameters using the eVOLVER electron app. This will be overwritten once the experiment code starts running, but can be helpful for preheating media or trying out different stir rates.
10. If stir bars are jumping around (audibly) then the stir rate may be too high for the volume of culture you are using. Commonly used values are between 8 and 12.

## Relevant Forum Posts

[Recommendations for Septas and Needles?](https://www.evolver.bio/t/recommendations-for-septas-and-needles/59)

[Using nylon caps to determine culture volumes](https://www.evolver.bio/t/using-nylon-caps-to-determine-culture-volumes/91)

[Making vial caps w/ laser cutter and pipette tip](https://www.evolver.bio/t/making-vial-caps-w-laser-cutter-and-pipette-tip/67)

