Team:GeorgiaTech/Electrocompetent Cells Protocol

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==Electrocompetent Cells Protocol==
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# Grow media culture of the cells at 37 degrees
# Grow media culture of the cells at 37 degrees
# Inoculate flask with 500mL of SOB media with 1mL of the media culture
# Inoculate flask with 500mL of SOB media with 1mL of the media culture
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# Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees
# Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees
# Decant the supernatant and resuspend each pellet in 50 mL of ice cold ddH2O.
# Decant the supernatant and resuspend each pellet in 50 mL of ice cold ddH2O.
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# Combine resuspensions into 2 centrifuge bottles . Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees. At this step,
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# Combine resuspensions into 2 centrifuge bottles . Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees. At this step, rinse two 50 mL conical tubes with ddH2O and chill on ice.
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rinse two 50 mL conical tubes with ddH2O and chill on ice.
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# Decant the supernatant and resuspend each pellet in 20 mL of ice cold 10% glycerol. Transfer each suspension to a 50 mL conical tube.
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# Decant the supernatant and resuspend each pellet in 20 mL of ice cold 10%
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# Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
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glycerol. Transfer each suspension to a 50 mL conical tube.
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# Carefully aspirate the supernatant with a sterile Pasteur pipette (pellets lose adherence in 10% glycerol). Resuspend each pellet in 1 mL of ice cold 10% glycerol by gently swirling. The final OD600 of the resuspended cells should be 200-250.
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# Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees. Start putting 1.5 mL microfuge tubes on ice if not
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# Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer.
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already chilled.
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# Carefully aspirate the supernatant with a sterile Pasteur pipette (pellets lose
+
-
adherence in 10% glycerol). Resuspend each pellet in 1 mL of ice cold 10%
+
-
glycerol by gently swirling. The final OD600 of the resuspended cells should be ~
+
-
200-250.
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# Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen.
+
-
Store frozen cells in the -80°C freezer.
+

Latest revision as of 01:58, 28 September 2013

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Electrocompetent Cells Protocol

  1. Grow media culture of the cells at 37 degrees
  2. Inoculate flask with 500mL of SOB media with 1mL of the media culture
  3. Place flask in the 37 degree Celsius shaker.
  4. Check the OD reading about every hour
  5. Once the OD reaches between 0.3 and 0.4, the rest of the protocol can be followed.
  6. Put the flask on ice
  7. Split the contents in the flask into 2 250mL centrifuge tubes
  8. Centrifuge at 1000 RCF, 3 Decel, 9 Accel, 4 degrees. Make sure to adjust the rotor setting.
  9. Decant the supernatant and resuspend each pellet in 100 mL of ice cold ddH2O.
  10. Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees
  11. Decant the supernatant and resuspend each pellet in 50 mL of ice cold ddH2O.
  12. Combine resuspensions into 2 centrifuge bottles . Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees. At this step, rinse two 50 mL conical tubes with ddH2O and chill on ice.
  13. Decant the supernatant and resuspend each pellet in 20 mL of ice cold 10% glycerol. Transfer each suspension to a 50 mL conical tube.
  14. Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
  15. Carefully aspirate the supernatant with a sterile Pasteur pipette (pellets lose adherence in 10% glycerol). Resuspend each pellet in 1 mL of ice cold 10% glycerol by gently swirling. The final OD600 of the resuspended cells should be 200-250.
  16. Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer.
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