Difference between revisions of "Team:Uppsala/Experiments"

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<h3>Transformation on chip protocol:</h3>
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<h4>Preparing for cell transformation</h4>
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  <li>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)</li>
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  <li>Heat the chip by running water at 65°C  and 300mL h-1 through the heating channels.</li>
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  <li>Take out 50uL of CaCl2 competent cells from -80°C freezer and thaw them on ice for 5 min.</li>
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  <li>Take  10uL of competent cells into a separate tube for negative control.</li>
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<li>Add 0.8uL of DNA (at a concentration of approximately 70 ng uL-1 ) to the remaining 40uL of cells. Incubate cell and DNA suspension for 20 min on ice.</li>
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</ol>
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<h4>Running the transformation chips</h4>
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<li>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. </li>
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<li>For each transformation: pipette 6uL of cell and DNA suspension into the syringe connector on the chip.</li>
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<li>Push the suspension into the transformation channel by pushing air in with a syringe. </li>
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<li>Fill the transformation channel and heat shock the suspension for 45 sec. </li>
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<li>Push the cell suspension through and collect in an Eppendorf tube filled with 100uL SOB. </li>
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<li>Place the tube on ice immediately. </li>
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<li>Incubate for 1.5 h at 37°C and then spread on an agar plate with the appropriate antibiotic. </li>
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Revision as of 15:29, 15 October 2016


Parts

Transformation on chip protocol:

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 50uL of CaCl2 competent cells from -80°C freezer and thaw them on ice for 5 min.
  4. Take 10uL of competent cells into a separate tube for negative control.
  5. Add 0.8uL of DNA (at a concentration of approximately 70 ng uL-1 ) to the remaining 40uL of cells. Incubate cell and DNA suspension for 20 min on ice.

Running the transformation chips

  • 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.
  • For each transformation: pipette 6uL of cell and DNA suspension into the syringe connector on the chip.
  • Push the suspension into the transformation channel by pushing air in with a syringe.
  • Fill the transformation channel and heat shock the suspension for 45 sec.
  • Push the cell suspension through and collect in an Eppendorf tube filled with 100uL SOB.
  • Place the tube on ice immediately.
  • Incubate for 1.5 h at 37°C and then spread on an agar plate with the appropriate antibiotic.