Team:Uppsala/Experiments


Experimental Procedures

Microfluidics

Transformation on chip:

Preparing for cell transformation

  1. Set up chip by connecting 0.583mm tubes insulated with paper towels to the heat channels. Use bent metal connectors. Connect tubes to two peristaltic pumps; one for each heating channel. Place a syringe needle in the inlet of the transformation channel and a tube in its outlet. (See image of setup)
  2. Heat the chip by running water at 65°C and 300mL h-1 through the heating channels.
  3. Take out 50µL of CaCl2 competent cells from -80°C freezer and thaw them on ice for 5 min.
  4. Take 10µL of competent cells into a separate tube for negative control.
  5. Add 0.8µL of DNA (at a concentration of approximately 70 ng µL-1 ) to the remaining 40µL of cells. Incubate cell and DNA suspension for 20 min on ice.
Running the transformation chips
  1. Flush the cell channel with 2 mL of 75% ethanol with a syringe. Dry the cell channel with air. This cleaning step should be repeated after each transformation to avoid contamination.
  2. For each transformation: pipette 6µL of cell and DNA suspension into the syringe connector on the chip.
  3. Push the suspension into the transformation channel by pushing air in with a syringe.
  4. Fill the transformation channel and heat shock the suspension for 45 sec.
  5. Push the cell suspension through and collect in an Eppendorf tube filled with 100µL SOB.
  6. Place the tube on ice immediately.
  7. Incubate for 1.5 h at 37°C and then spread on an agar plate with the appropriate antibiotic.

General

Most of the general lab work, such as assemblies, were performed following the protocols in Synthetic Biology: A lab manual.

Liljeruhm, J. Gullberg, E. Forster, AC. 2014. Synthetic Biology: A lab manual. World Scientific Publishing Co. Singapore

For the IMAC purification we used the BioRAD IMAC Instruction Manual as a source for most of our work.