Making competent cells

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Chemically Competent Cells

  1. Streak cells onto LB overnight
  2. grow single colony in 15mL LB overnight
  3. take 1mL of overnight culture and Grow cells to OD=0.5 in 125mL of LB shaken for 4hrs-ish at 37C 200rpm 20mm orbit in a 250mL erlenmeyer flask with foam plug
  4. spin down at 3500rpm for 5 min in 8 15mL tubes full up.
  5. Decant completely, remove all the excess liquid with a pipette (very important to do so but do not disturb the pellet).
    6-8 should be done on ice and done as quickly as possible:
    1. Resuspend one of the 8 tubes with 1mL cold LB
    2. take the entire resuspension into another spun down tube
    3. use the liquid from the previous resuspension to resuspend the second tube
    4. Repeat until all the pellets are resuspended and concentrated into one tube. Resulting suspension should be about 1.2mL. Keep all tubes in contact with ice while resuspending.
  6. Add that volume of ice cold TSS 2x to the tube and mix gently by pipetting up and down.
  7. Dispense 100uL into sterile prechilled pcr tube. Makes about 24 reactions worth.

Can potentially be stored at -20C for about 3 weeks with just enough transformation efficiency to be useful. Source.


TSS Buffer:

  1. 5g PEG 8000
  2. 1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H20)
  3. 2.5 mL DMSO
  4. Add LB to 50 mL