Team:GeorgiaTech/Chemically Competent Transformation

From 2013.igem.org

Revision as of 00:41, 28 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:

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.