Making Competent Cells

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Day 1

Restreak the bacteria on LB plates with appropriate antibiotics from a -80 stock. It is best not to restreak from a competent cell stock.

Day 2

  1. Grow up a single colony in 10mL of LB+antibiotic
  2. Autoclave large centrifuge bottles
  3. Prepare 1L LB in a flask
  4. Prepare the TSS solution (if there is none in the gel plate fridge):

Autoclave individually:

85mL LB
10g PEG-3350 in 5mL H2O (other PEG sizes are ok)
2mL 1M MgCl2
Allow those to cool, then combine, filter sterilize and fridge

Day 3

  1. Add 10mL overnight culture to pre-warmed 1L LB
  2. Clean out and prechill the rotor on ice
  3. Prechill the centrifuge tubes and a large serological pipette
  4. Grow culture to OD=0.5, takes between 2-3 hours
  5. Shake on ice to stop the growth
  6. Spin cells at 3500rpm for 15min or 6500 for 5 minutes
  7. Resuspend the pellets in the 25-50ml LICE-COLD TSS
  8. Aliquot into 200uL tube using multipipetter in the cold room
  9. Freeze cells in liquid nitrogen bath.


25-50mL pipette for cell suspension
500mL centrifuge bottles
whole beaker of eppendorf tubes
5mL multipipette
TSS solution


  1. Grow cells in ~4 mL LB until cloudy (OD600=0.5)
  2. Put on ice
  3. Transfer 1mL into an eppendorf tube on ice, let cool
  4. Centrifuge full speed for 30 sec, toss out supernatant
  5. Resuspend in 90uL of TSS solution
  6. Add 10uL KCM
 Step 6.5:  do the negative control before adding the DNA
  1. Add 1uL plasmid DNA
  2. Let sit on ice for 10min, heat shock 90 sec at 42, ice for a minute,

rescue 1 hr, then incubate and/or plate

        • Always do negative controls on your competent cell prep!

Sector a region of your plate and spread about 5uL of untransformed competent cells to confirm that they are free of contamination.