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Team:GeorgiaTech/Chemically Competent Transformation
From 2013.igem.org
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*Add '''2 µL''' of purified plasmid | *Add '''2 µL''' of purified plasmid | ||
*Incubate the PCR tube in the PCR machine with HEATSHOK program: | *Incubate the PCR tube in the PCR machine with HEATSHOK program: | ||
| - | 4°C for 10 min | + | ===Heat Shock protocol for PCR machine=== |
| - | 42°C for 30 sec | + | *4°C for '''10 min''' |
| - | Hold at 4°C until we add 222 µL of SOC | + | *42°C for '''30 sec''' |
| - | 37°C for 1hr | + | *Hold at 4°C until we add '''222 µL''' of SOC |
| - | Holds at 4°C until we are ready for the next step. | + | *37°C for '''1hr''' |
| - | + | *Holds at 4°C until we are ready for the next step. | |
| - | + | ====Estimated Time: 1 hr 11 mins==== | |
| + | |||
**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 20:30, 27 September 2013
Contents |
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:
Heat Shock protocol for PCR machine
- 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.
Estimated Time: 1 hr 11 mins
- 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.
