Making Competent Cells
Restreak the bacteria on LB plates with appropriate antibiotics from a -80 stock. It is best not to restreak from a competent cell stock.
- Grow up a single colony in 10mL of LB+antibiotic
- Autoclave large centrifuge bottles
- Prepare 1L LB in a flask
- Prepare the TSS solution:
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
- Add 10mL overnight culture to pre-warmed 1L LB
- Clean out and prechill the rotor on ice
- Prechill the centrifuge tubes and a large serological pipette
- Grow culture to OD=0.5, takes between 2-3 hours
- Shake on ice to stop the growth
- Spin cells at 3500rpm for 15min or 6500 for 5 minutes
- Resuspend the pellets in the 25-50ml LICE-COLD TSS
- Aliquot into 200uL tube using multipipetter in the cold room
- Freeze cells in liquid nitrogen bath.
rotor 25-50mL pipette for cell suspension 500mL centrifuge bottles whole beaker of eppendorf tubes 5mL multipipette TSS solution
- Grow cells in ~4 mL LB until cloudy (OD600=0.5)
- Put on ice
- Transfer 1mL into an eppendorf tube on ice, let cool
- Centrifuge full speed for 30 sec, toss out supernatant
- Resuspend in 90uL of TSS solution
- Add 10uL KCM
Step 6.5: do the negative control before adding the DNA
- Add 1uL plasmid DNA
- 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.