Team:MIT mmethods

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Revision as of 02:10, 15 October 2010

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
Material
  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.

@@ Line 54: / Line 54: @@
      &nbsp;&nbsp;&nbsp;&nbsp;b. De-gas for 20 minutes<br>
      &nbsp;&nbsp;&nbsp;&nbsp;Note: Open/close valve quickly to get rid of bubbles faster. When finished, close vacuum; open valve slowly. <br>
-<wiki>Procedure
-#&nbsp;Pour body liquid in a plastic cup while measuring its weight (Make sure to cover the balance with tissue)
-#Reset scale, pour curing agent while measuring its weight (in 1:10 ratio to body liquid)
-#Stir the mixture
-#Remove the bubbles in the vacuum chamber
-&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;a. Turn on vacuum and close the valve for sustaining vacuum (Make sure to cover the chamber with tissue) - Note: Lift container to be sure &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; the vacuum is on.
-&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;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  ==
-#Blow the wafers with air, clear of all debris
-#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.
-#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  ==
-#Bake the poured wafers for 4-8 hours at 80C (recommended: 24hours to cross-link most of PDMS monomers)&nbsp;
-#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 ==
-#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.
-#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.
-#Put devices in beaker with DI water, autoclave (wet / dry, 20 / 10 minutes)
-#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)
-#Dry in oven overnight @ 80C.</wiki>
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