Team:Macquarie Australia/WetLab/Protocols/CompetentCells

Making Competent Cells

• Using a sterile plastic loop, pick 10-12 large (2-3mm in diameter) colonies from the plate. Inoculate to 150mL of SOB medium in a 1L flask, and grow overnight at 18-22^oC, 200-250rpm.
• A600 should be 0.2-0.8 to harvest. Preferably, cells should be in mid log phase with A600 ~ 0.5.
• Remove the flask from the incubator and place on ice for 10 minutes.
• Transfer the culture to a 15mL centrifuge tube and spin at 2500 x g for 10 min at 4^oC
• Pour off and discard the supernatant, and immediately place the tube on ice.
• Resuspend your cells in 1mL of ice-cold TB buffer, make sure there are no clumps of cells left, but also treat your cells gently and keep them cold.
• Add ice-cold TB buffer to bring volume up to 1/5th of the original culture volume (~30mL in this case). Mix the tube by gently inverting 3 times.
• Incubate the tube on ice for 10 minutes.
• Centrifuge at 2,500 x g for 7 minutes at 4^oC, discard the supernatant.
• Gently resuspend the cells in ~1/20th of the original culture volume of ice-cold TB buffer. NOTE: 1/20th is based on and OD600 of 0.5, so adjust volume accordingly. e.g. if the culture OD600 was 0.1 then resuspend in 1/100th of original volume.
• Pre-chill 1.5ml Eppendorf tubes on ice. Add 930µl of your cell suspension, keeping the remainder on ice in the 15mL tube.
• Add 70µl of DMSO to the 930µl of cell suspension. Mix gently by swirling, and place on ice.
• Aliquot 100µl of the competent cell/DMSO mixture into fresh microcentrifuge tubes. Label the tubes with: Date – Strain. Snap freeze with liquid nitrogen.
• Repeat step 11-13 for the rest of your cell suspension in step 10. Store cells at -80^oC.

Buffer Preparation

TB BUFFER

• Ingredients: 3g PIPES, 10.9g MnCl2-4H2O, 2.0 g CaCl2-2H2O, 18.6 g KCl.
• Methods: All components (except for MnCl2-4H2O) were mixed and dissolved in 500 mL of water and pH adjusted to 6.7 with KOH. Then, MnCl2-4H2O, was dissolved in 300 mL of water, mixed and solution adjusted to 1 L. Sterilisation via filtration followed through a pre-rinsed 0.45 µm filter unit and stored at 4°C.

EDTA BUFFER

• Ingredients: 37.22g EDTA solid, 180 mL of water and pH adjusted to 8.0 using 10M NaOH.
• Methods: Components were combined then pH adjusted.

TAE BUFFER

• Ingredients: 121.2g Tris base (dissolved in water) with 28.55mL of glacial acetic acid & 50mL 0.5M EDTA (pH 8.0).
• Methods: A total volume of 500 mL was made up as a 50x stock solution using all components.