Team:GeorgiaTech/Chemically Competent Transformation

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(Difference between revisions)
(Created page with "===Materials=== *Chemically Competent Cells -'''20 µL''' *PCR machine *10% BME ( beta mercaptoethanol ) - '''0.88 µL''' *SOC media - '''222 µL''' *Pipette tips with cut ends (...")
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*Incubate on ice for '''10 min'''
*Incubate on ice for '''10 min'''
*Add '''2 µL''' of purified plasmid
*Add '''2 µL''' of purified plasmid
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*Incubate the PCR tube in the PCR machine with HEATSHOK program in the lab 0245. See [[HEATSHOK Protocol|HEATSHOK protocol]] [[HEATSHOK_Potocol|'''Here''']].
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*Incubate the PCR tube in the PCR machine with HEATSHOK program:
 +
4°C for 10 min
 +
42°C for 30 sec
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Hold at 4°C until we add 222 µL of SOC
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37°C for 1hr
 +
Holds at 4°C until we are ready for the next step.
 +
[edit]Estimated Time: 1 hr 11 mins
*Add '''222 µL''' of SOC to fill to a total volume of '''250 µL''' when the PCR machine is holding at '''4°C'''
*Add '''222 µL''' of SOC to fill to a total volume of '''250 µL''' when the PCR machine is holding at '''4°C'''
**Be sure to sterilize the bottle with EtOH, and not contaminate the media when loading it into the heat cycler
**Be sure to sterilize the bottle with EtOH, and not contaminate the media when loading it into the heat cycler
*Plate '''20 µL''' and '''200 µL''' from each tube using the spreaders on [[LB]]/agar [[plates]] with the appropriate antibiotic
*Plate '''20 µL''' and '''200 µL''' from each tube using the spreaders on [[LB]]/agar [[plates]] with the appropriate antibiotic
*Leave [[plates]] at '''37°C''' overnight.
*Leave [[plates]] at '''37°C''' overnight.

Revision as of 20:29, 27 September 2013

Materials

  • Chemically Competent Cells -20 µL
  • PCR machine
  • 10% BME ( beta mercaptoethanol ) - 0.88 µL
  • SOC media - 222 µL
  • Pipette tips with cut ends ( To decrease the amount of cells destroyed during the transferring and resuspending procedure)
  • Plates with appropriate antibiotic (if stored in fridge, place in incubator before doing the transformation so that the plates are warm when it's time to plate)

Procedure

  • Transfer 20 µL chemically competent cells from -80°C freezer to a PCR tube using a cut pipette tip
  • Thaw competent cells over ice --> They should NOT be taken from above 4°C freezers)
  • Thaw 10% BME - 0.88 µL (Must be completely thawed)
  • Add 10% BME to the PCR tube with the competent cells
  • Incubate on ice for 10 min
  • Add 2 µL of purified plasmid
  • Incubate the PCR tube in the PCR machine with HEATSHOK program:

4°C for 10 min 42°C for 30 sec Hold at 4°C until we add 222 µL of SOC 37°C for 1hr Holds at 4°C until we are ready for the next step. [edit]Estimated Time: 1 hr 11 mins

  • Add 222 µL of SOC to fill to a total volume of 250 µL when the PCR machine is holding at 4°C
    • Be sure to sterilize the bottle with EtOH, and not contaminate the media when loading it into the heat cycler
  • Plate 20 µL and 200 µL from each tube using the spreaders on LB/agar plates with the appropriate antibiotic
  • Leave plates at 37°C overnight.
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