Team:MIT mmethods

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Bacterial Construction Protocol
Bacterial Experimental Protocol
Phage western blot
Mammalian Protocol
Microfluidic stress

Materials

mammalian methods

The Mammalian team focused on creating a pressure-sensitive promoter and creating a standard protocol for mammalian genes.

1 HTD Preparation Protocol
    1.1 PDMS Mixture Preparation
    1.2 PDMS Pouring
    1.3 PDMS Baking
    1.4 PDMS-Device punching and Bonding
    1.5 PDMS Device Bonding
    1.6 PDL coating
    1.7 Collagen filling
    1.8 Cell Seeding
2 Protocol for Deflection Experiments
    2.1 Tubing Setup Details
    2.2 Adding Medium to Channels
    2.3 Connecting device to pressure valve
    2.4 Microcontroller details
HTD Preparation Protocols (adapted from Yannis, Alisha)

PDMS Mixture Preparation
Materials
  • 1. PDMS Body liquid (10 parts)
  • 2. PDMS curing agent (1 part)
  • 3. Negative pattern wafer
  • 4. Microscope slides (1mm thick)

Procedure
  • 1. Pour body liquid in a plastic cup while measuring its weight (cover the balance with tissue)
  • 2. Reset scale, pour curing agent while measuring its weight (in 1:10 ratio to body liquid)
  • 3. Stir the mixture
  • 4. Remove the bubbles in the vacuum chamber
      • a. Turn on vacuum and close the valve for sustaining vacuum (cover the chamber with tissue)
      Note: Lift container to be sure the vacuum is on.
      b. De-gas for 20 minutes
      Note: Open/close valve quickly to get rid of bubbles faster. When finished, close vacuum; open valve slowly.

PDMS Pouring
  • 1. Blow the wafers with air, clear of all debris
  • 2. Pour the PDMS onto the wafers. Make sure there is enough PDMS to completely fill the hole cut in the old PDMS and does not form a meniscus. Note that any devices that are not perfectly flat on top cannot be used with a syringe pump.
  • 3. Let PDMS on wafer stand for 15-30mins, then blow air bubbles that have risen to the surface and put in oven. Do NOT de-gas wafer. This has lead to cracking and we only have the one.

    • PDMS Baking
    • 1. Bake the poured wafers for 4-8 hours at 80C (recommended: 24hours to cross-link most of PDMS monomers)
    • 2. Detach by cutting with razor (carefully and slowly peel it off, starting from the edges and going in the circumferential direction, in order to avoid tearing posts apart)


      • PDMS-Device punching and Bonding
    • 1. Cut devices apart using a razor and the outline on the mask. Punch 4mm holes in the cell seeding and reservoir ports, a 2mm hole in the outlet port, and use the syringe to punch the gel filling ports.
    • 2. Clean devices with transparent tape. It’s better to punch the devices off of the paper covered acrylic because the paper makes the devices very dirty.
    • 3. Put devices in beaker with DI water, autoclave (wet / dry, 20 / 10 minutes)
    • 4. Remove devices from beaker and place devices in empty pipette tip box, up to 6 per box. Autoclave again in box (switch setting to dry)
    • 5. Dry in oven overnight @ 80C.

      • PDMS Device Bonding - Plasma Treatment
        1. Clean area and all tools with ethanol
        2. Place one device and one coverslip per platform, up to 3 platforms (platform looks like a small glass slide)
        3. Close the door (turn black wheel until it can't turn any more) and valve, confirm seal, and turn on vacuum (“Pump” switch) for 2 minutes.
        4. 1min and 40 sec plasma treatment: Turn “Power” switch on, turn radiation dial to low, medium, then high. After observing a purple glow, slowly;open the valve (black wheel on the door, spin backwards slowly, fractions of a turn!) until the glow becomes bright.
        5. Wait for 1 minute 30 seconds
        6. Turn off Power->RF->Pump, unscrew black wheel, and take out devices
        7. Bring the devices and glass coverslip into contact with tweezers. Apply firm pressure with your fingers from one side to the other, avoiding air gaps between the coverslip and the device.
        Note: When making 2-layer devices, after bonding the two layers together, bake in the oven overnight. Then punch holes for the top layer and autoclave. If desired, can fill devices with 60 ul of media after bonding, and proceed directly to the cell seeding step

      PDL Coating
        a. Fill the devices with PDL (~100-150µL/device) b. Wash twice with water after 24 hours. Make sure that all regions are washed, in order to avoid heterogeneous surface coating. If you get bubbles, try to remove them by using the pipette applying suction.

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