Making Competent Cells

LARGE SCALE

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

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.

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

SMALL SCALE

Step 6.5: do the negative control before adding the DNA rescue 1 hr, then incubate and/or plate
 * 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
 * 1) Add 1uL plasmid DNA
 * 2) Let sit on ice for 10min, heat shock 90 sec at 42, ice for a minute,


 * 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.