Difference between revisions of "Team:Uppsala/Experiments"

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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>
 
   <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>
 
   <li>Heat the chip by running water at 65°C  and 300mL h-1 through the heating channels.</li>
 
   <li>Heat the chip by running water at 65°C  and 300mL h-1 through the heating channels.</li>
−
   <li>Take out 50uL of CaCl2 competent cells from -80°C freezer and thaw them on ice for 5 min.</li>
+
   <li>Take out 50µL of CaCl2 competent cells from -80°C freezer and thaw them on ice for 5 min.</li>
−
   <li>Take  10uL of competent cells into a separate tube for negative control.</li>
+
   <li>Take  10µL of competent cells into a separate tube for negative control.</li>
−
<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>
+
<li>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.</li>
 
</ol>
 
</ol>
  
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<ol type="1">
 
<ol type="1">
 
<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>
 
<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>
−
<li>For each transformation: pipette 6uL of cell and DNA suspension into the syringe connector on the chip.</li>
+
<li>For each transformation: pipette 6µL of cell and DNA suspension into the syringe connector on the chip.</li>
 
<li>Push the suspension into the transformation channel by pushing air in with a syringe. </li>
 
<li>Push the suspension into the transformation channel by pushing air in with a syringe. </li>
 
<li>Fill the transformation channel and heat shock the suspension for 45 sec. </li>
 
<li>Fill the transformation channel and heat shock the suspension for 45 sec. </li>
−
<li>Push the cell suspension through and collect in an Eppendorf tube filled with 100uL SOB. </li>
+
<li>Push the cell suspension through and collect in an Eppendorf tube filled with 100µL SOB. </li>
 
<li>Place the tube on ice immediately. </li>
 
<li>Place the tube on ice immediately. </li>
 
<li>Incubate for 1.5 h at 37°C and then spread on an agar plate with the appropriate antibiotic. </li>
 
<li>Incubate for 1.5 h at 37°C and then spread on an agar plate with the appropriate antibiotic. </li>

Revision as of 06:39, 16 October 2016


Experimental Procedures

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 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.