5 and 7 Port Nylon Tubing Caps Construction Protocol

Materials
Semi-clear white hard nylon tubing (1/8" OD)
White flexible silicone rubber tubing (3/32" ID (inner diameter))
Blue flexible silicone rubber tubing (3/32" ID (inner diameter))
Five 3/32" female barb to luer connectors
Mixing tray (here a small weighboat)
Disposable applicator (here a wooden stir stick; pipette tip also works)
[Optional - for color coding ports] Red flexible silicone rubber tubing (3/32" ID (inner diameter))
[Optional - for color coding ports] Black flexible silicone rubber tubing (3/32" ID (inner diameter))
[Optional] 3D printed (SLS, nylon PA-12, vapor smoothed) bubbler for in-vial aeration

Design
Choose the minimum number of ports necessary
You will need to block up ports that are unused if you want to do emergency efflux
Therefore, choose the cap with the minimum number of ports that you need
For example, counting ports for PACE
Lagoon: 1 vial-to-vial + 1 efflux + 2 inducers + 1 emergency efflux = 5 ports
Cell reservoir: 1 media influx + 1 efflux + 1 vial-to-vial + 1 emergency efflux = 4 ports
For simplicity, you may want to just use this type of cap and plug the extra port with a luer cap.
Efflux straw sets the volume of the culture
Increase influx accuracy
Important for low volume influx like inducers
Angled influx tubing should touch the inside of the glass vial to create a stream of liquid rather than drops.
It should also be well above the efflux line to avoid contamination.
[Optional] Use different colors of tubing to denote different ports (see above)
Construction Protocol
0. Mark fluid lines on a glass eVOLVER vial (see above image)
This will be used to set the efflux tube height, which sets the volume in the eVOLVER vial.
Tape an eVOLVER vial
Using a serological pipette, measure water into the vial in increments of 5 mL
Mark the volume at each increment on the tape at the bottom of the meniscus
1. Cut Nylon Tubing to Various Lengths Needed

Cut the necessary components of the hard semi-clear white nylon tubing (1/8" OD). Refer to the table below for lengths.
The table below has the necessary components for the construction of one cap.
1 x Regular Efflux (culture volume of 25 mL)
3 inches
1 x Emergency Efflux
1.5 inches
2 x Influx
2.5 inches
1 x Gas Bubbler
2 inches
Cut the necessary components of the flexible nylon tubing. Refer to the table below for lengths.
The table below has the necessary components for the construction of one cap.
1 x Regular Efflux
1.5 inches
Blue
1 x Emergency Efflux
1.5 inches
Black
2 x Influx
1.5 inches
Clear
1 x Gas Bubbler
1.5 inches
Red
1 x Bubbler Connector
5/8 inches
Clear
2. Assemble Cap
When handling epoxy, use gloves to avoid getting it on skin as it can eventually lead to allergic reactions.
Steps
You may need to drill out the ports in the 3D printed vial cap if powder is blocking them.
Insert the hard semi-clear white nylon tubing (1/8" OD) into each corresponding port in the cap as per the image below.

Screw the cap into the glass vial with measuring tape on the side and ensure that:

Follow the epoxy directions to mix up a small amount of epoxy in a disposable dish. Use even pressure to get even amounts of resin and hardener. Use a toothpick or other similarly sized applicator to put the minimum amount of epoxy around the outside area of the cap where the ports and the hard nylon tubing connect, ensuring to spread the epoxy in between any gaps.
Set assembled caps upright (with a vial rack or spare vials) so that epoxy doesn't drip into the sampling port as it cures.
Set cap on its side to cure overnight.
Next day, add the appropriate color flexible nylon tubing (3/32" ID) to each hard nylon tubing (1/8" OD) as per the table above.
Color flexible tubing connected to cap. [Bubbler Only] Connect the flexible nylon tubing (3/32" ID) bubbler connector component onto the inside end of the gas bubbler hard tubing. Allow the tubing to reach halfway through the connector tubing.
[Bubbler Only] Connect the constructed gas bubbler onto the other side of the connector tubing. The end of the gas bubbler hard tubing that is inserted in the cap and the gas bubbler should connect as per image below.
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