Running the eVOLVER
Updated 8/16/2018 07:49 PM
Introduction
Materials
- eVOLVER
- vials, etc
- YPD or SDC media
- in eVOLVER-friendly bottles
- 1L 10% bleach
- 1L 70% ethanol
- 15mL of grown cells
Procedure
Pre-eVOLVER Prep
- Prep media
YPD - fill dishwashed bottles and autoclave w/ media in them, add sugar source after
SDC - sterile filter in autoclaved eVOLVER bottles
- Morning of, autoclave vials w/ stir tiny bars in them
a) Check the tubing on the caps to make sure they're clear / push some DI water through them prior to autoclaving to wash out any residue, make a couple extra vials just in case.
b) Vials face down have been dishwashed and autoclaved
- Ensure that the vials are clean at the bottom
(where OD is measured)
- Make 1L 10% bleach, and 1L 70% Ethanol
eVOLVER
- Inspect fluidic lines for leftover fluid or salt residue
- Inspect fluidic connections, make sure all are connected
- Turn on (5V then 12V)
- Find the correct fluidic splitter for the setup from drawer, and connect input (clear) lines - making sure there are no leaks
- Using the tubing connectors hook up input lines to 10% bleach and crack lid for airflow
- Place other ends of lines & output lines in a large (2+L) beaker
- add 100-200mL bleach from your 10% stock to the beaker
- Add 1.5-2L bleach into large empty waste container
- Using MatLab, Check IP address (192.168.1.?), and run pumps 1:32 (can do less if not using all vials) for 1 second to confirm that the proper evolver is receiving the message
- If yes, then up time to 5-7 seconds and run several times until all fluid lines are filled with bleach
- While running this, ensure that all pumps are firing that should be and that the lines fill with bleach
- Ensure that the output pumps are working as expected (push them down into the large and that those lines get filled with bleach
- Let sit ~30min to sterilize lines
- Run pumps in 10-20 sec increments until beaker empties most of the bleach to the waste
- Repeat the bleach process, but with 70% ethanol
- Rinse out both input and output lines with ethanol to remove bleach residues
Ethanol kills immediately, no need to wait 30 min contact time
- Following the ethanol, attach media and run pumps until a few drops of media is coming out the input lines (flushed the ethanol out) while letting output lines pick up some air so that ethanol does not drip back to the vials
- At this stage, insert the vials into the smartsleeves, and hook-up the lines to the proper positions (utilize the colored strips).
Output lines get attached to longer tube in the cap (the pipette type one), input get attached to the short flexible one
- Check the temperature of the vials by feel: if any of them are overly hot, run the temp setting code in Matlab at 500 (comment out everything else)
- Run either the input lines or both sets and fill the vials with media (~30mL total in 5mL increments). Ensure that the output lines are properly picking up media so that no overflows occur
For code:
- From the “Experiment” folder pull up the “main” code. Near the bottom of the file, change the experiment name.
- In ??? file ensure that the temperature is set as desired
a) Normally 30 for yeast, 37 for E. coli
b) setting found about 3/4 of the way to the bottom
c) Also a good time to manually check the heat of the vials
- In ??? file ensure that the proper OD ranges are set for the respective vials used
9999 for the unused ones
- Pull up a command window, type cd and drag the experiment folder w/ all the needed files into the command window
- Start typing “main” till the window autofills in with the desired code. and hit enter
- Start experiment and answer the questions
don't calibrate to blank, and don't start from a previous experiment, turn off pumps is at your discretion
- In the data files ensure that the OD reading's are within reason, and the temperatures are either at desired level or progressing towards it
- If temp and OD readings are good, start the experiment again and calibrate the OD to blank
- Check the OD's once calibrated to blank to ensure that they're actually sitting right about 0
Prepare to inoculate the cells:
- Prior to inoculating, measure OD of the cultures and calculate how much to add in order to produce a culture at desired starting OD
- Measured OD/Desired OD = fold dilution
- 30mL (vial volume) / fold dilution = mL cells to add
- Then inoculate the vials, and ensure that the cultures are somewhat close tot he desired OD. If so..... then we are good to go.
Take Down:
- Prepare 1L 10% bleach and 1L 70% ethanol
- Turn off experiment by closing the experiment window
- To avoid chances of flooding, remove vials and place in the white autoclave rack
- Using a connector, attach lines to 10% bleach
- Using Matlab (Double check IP address), run pumps for 1 second to ensure signal is being sent to proper location
- Run short bursts of bleach (5s) to ensure that the waste lines are still picking up up media
- Up the time (10-20s) and run bleach through vials until they all appear cleared of media
- Let sit 30min
- Repeat with ethanol until vials appeared cleared of bleach
- Detach lines from vials and run pumps for a couple seconds to fill them with some air
- Shut off the evolver (12V first, then 5v)
- Rinse out the vials with DI water (being careful not to lose the stir bars) and return them to the carboard box facing up. Vials are dish-washed in a large batch
- Let caps rinse in DI water for some time (overnight works)
- Rinse media bottles out prior to dish-washing, push some DI water through the lines to ensure all the media is out
This is the general eVOLVER protocol for set up, take down, etc. as taken from FynchBio's internal wiki on August 16, 2018. Originally written by Kole Sedlack.