Transformation of chemically competent bacteria

This protocol is based on the transformation protocol laid out in Sambrook and Russell. Transformed strains are only stable on plates for 7-10 days, therefore glycerol stocks or fresh transformations are required.

- Note: Due to the presence of recA and other DNA repair machinery in JS006 and derivatives, plasmids in these strains are less stable than in cloning strains such as DH10B. Therefore, glycerol stocks of networks in JS006 or derivative strains are less stable, and not a recommended means of maintaining these strains. Fresh transformations are a better option.

  1. Remove a 100µl aliquot of competent cells from the -80°C freezer
  2. Let the aliquot thaw on ice for 5-10 minutes, or until all ice crystals have melted
  3. Add 100µl of cells to a pre-chilled 14ml polypropelene tube on ice.
  4. Add 1µl (~50-100ng) pf plasmid DNA to the cells and mix gently.
  5. Incubate on ice for 30 min.
  6. Place at 42°C for 45 seconds.
  7. Incubate on ice for 2 min.
  8. Add 250µl of SOC media to the tube (See Protocols for recipe).
  9. Incubate at 37°C with shaking for 1 hour
    • Note: This step can be shortened or even omitted entirely if the plasmid used contains an ampicillin resistance cassette, but is required for other selective markers
  10. Plate 250µl of cell mixture on LB/agar plates with appropriate antibiotic(s).
  11. Incubate at 37°C overnight.

Scope Run Setup

This protocol is based on the protocol from Ferry et al. (2011).

Cell Culture and Media

  1. Pass an overnight culture of the strain to be analyzed at a 1:1000 dilution into fresh media (at least 10ml) in an Erlenmeyer flask.
  2. Incubate the culture at 37°C with shaking until OD600 reaches 0.1-0.2, usually 3-4 hours for JS006-based strains.
  3. Transfer 25ml of 2x LB media to a 50ml conical tube
  4. Add inducers and antibiotics, such that they are at 1x concentration for 50ml of media.
  5. Fill to the 50ml mark on the conical tube with sterile DI H2O
    • Note: Wrap the conical tube in foil and store at 4°C for up to 7 days. IPTG and other sugars are photosensitive, storage without foil will lead to a breakdown of inducers and a change in inducer concentration.

Warming and flushing the device

  1. Place 4-5 devices in a petri dish, and place in the incubator (usually 37°C for bacteria). Let the devices equilibrate for 15-30 minutes.
  2. Prepare 0.1% Tween-20 by adding 1ml of 1% Tween-20 to a 15ml conical tube. Add 9ml of sterile H2O to the tube.
  3. Sterile filter the 0.1% Tween-20 solution
  4. Transfer the sterilized solution to a 10ml syringe
  5. Top the syringe with a clean luer stub
  6. Attach a 6-8'' length of microbore tubing to the luer stub and insert a straight pin.
  7. Mount a chip to the stage insert and change the objective of the scope to 10x objective
  8. Beginning with the first port (determined by the specific design of the device being used), apply a small drop of 0.1% Tween-20 solution on the opening of the port.
  9. Insert the pin into the port, being careful to insert the pin vertically without cutting into the PDMS.
  10. Apply gentle pressure with the syringe, watching to ensure the trapping region of the chip flushes fully and is relatively clear of debris.
  11. Once the port is free of bubbles, carefully remove the pin without introducing any other bubbles into the device.
  12. Repeat steps 8 to 11 for all of the remaining ports
  13. Once all ports are flushed, top the chip with a large drop of flushing solution that covers all ports
  14. Leave the chip mounted to the scope until the cells are ready

Preparation of syringes

  1. Cut microbore tubing into 48'' lengths, one per port. If a dial-a-wave device is used, 96" lines are necessary to compensate for the increased height of the linear actuators
  2. Remove the plunger from a 10ml syringe
    • Note: For dial-a-wave devices, 25ml or 60ml syringes are used for the media reservoirs. The protocol is the same, but the larger syringes are required to reduce the impact of media depletion.
  3. Attach a luer stub to the syringe body
  4. Insert the luer stub into one end of the microbore tubing
  5. Insert a straight pin into the other end of the microbore tub
  6. Decant 10ml of sterile water into the syringe, letting it run down the side of the syringe body
  7. Flick the luer stub to clear any bubbles
  8. Let the water flow through the tube until all of the bubbles have been cleared
  9. Top the syringe with a square of parafilm
  10. Use the pin to poke a hole in the parafilm, and store the tube until it is time to load the chip
  11. Repeat steps 2 to 10 for remaining water syringes and media syringe(s)
  12. Once the cells have reached OD600 of ~0.1, transfer 10ml of cells to a 15ml conical tube. Spin at ~1500xg (3500RPM in a Sorvall SS-34 rotor) for 5 minutes.
  13. While the cells are spinning down, turn on the fluorescence source for the microscope so that the bulb can reach operating temperature.
    • Note: During the first 5-10 minutes of on-time, the intensity of the fluorescence source will fluctuate until the bulb reaches operating temperature. Therefore, it is important to allow a sufficiently long warm up time. Once the source is on, leave the output at 0% to prevent accidental exposure when configuring the run.
  14. Decant and discard the supernatant. Add 10ml fresh LB media with antibiotics and resuspend the cells by vortexing briefly
  15. Repeat steps 2 to 10 with the cell syringe

Loading the device and starting the experiment

Syringe Height Sheet

Flushing order for dual iDaw device

  1. Mount syringes to the carriers on the syringe tower and adjust the heights for loading
  2. Beginning with the media port and finishing with the cell port, insert the pin from the corresponding syringe into the port, making sure to insert the pin vertically without cutting into the PDMS
  3. After all pins have been inserted into the device, use a Kimwipe to remove the flushing solution from the top of the chip
  4. Gently gather the lines running into the chip and bend the lines slightly to prevent collision with the condenser. Affix the lines to the stage with tape
  5. Adjust the position of the stage until the trap is visible in the middle of the field of view
  6. Move the cell syringe up or down to modulate the speed of cells as they pass the trapping area
  7. Flick the cell line to force a single cell into the trapping area. Once a single cell has been trapped, stop flicking, as loading more than one cell can lead to premature trap filling
  8. Move the chip mount away from the objective and change to the 100x objective. Place a drop of microscopy oil on the 100x objective
  9. Replace the chip mount above the objective

  10. Focus the 100x objective

  11. Configure and test the autofocus
  12. Set the exposure doses for phase contrast and each fluorescence channel
  13. Set the fluorescence source to 10% output
  14. Set the time between exposures and set the destination directory for the data file. Name the file in a descriptive way.
  15. Once all configuration is done, lower the cell syringe to the running height and ensure that flow across the trapping area is at the correct rate and is from the media source.
  16. Turn off the room lights, close the door, start the run and turn off the computer monitor.
  17. After the first exposure, check to ensure that the autofocus is functioning and that the exposure times are correct.
  18. Leave the scope to run for the desired time length (usually 6-8 hours for bacteria)

Finishing the experiment

  1. Once the experiment has been running long enough for sufficient data to be acquired, finish the run using the scope software.
  2. Remove the chip from the mount and set it aside.
  3. Use 1/2 sheet lens paper to absorb most of the oil from the objective
  4. Place a drop of 70% ethanol (using the dropper bottle in the scope room) on the objective, and using the remaining 1/2 sheet of lens paper to absorb the ethanol
  5. Set the microscope objective to 10x in the software, then exit the software
  6. Turn off the fluorescence source
  7. Remove syringes from the tower
  8. Drain remaining solutions from the syringes and discard the syringe bodies
  9. Remove the luer stubs and the pins from the fluidic lines and discard the lines. Place the stubs and the pins in ethanol to be cleaned and reused.
  10. Place the chip in sharps waste

Export data from Nikon Elements

  1. Open run file in Elements
  2. File > ND Document > Export ND Document
  3. Select the correct destination to export
  4. Select TIF files with a mono image for each channel

 

Room 318 Microscope Components

 

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