Making the pSPIN-BAC E. coli Electrocompetent

I need to take the bacteria cells that already have the pSPIN-BAC and make them electrocompetent again so I can electroporate them with DiNV DNA. I am basing what I did on the large batch protocol layed out here.

20240123 Making Sterile Filtered 10% Glycerol

  • I need to make a total of 320mL, so I decided to make 500mL total
  • Combined 50mL of 100% glycerol in 450mL of molecular grade water in the top of a sterile filter unit from the BioStore (500mL capacity)
  • Used the serological pipette tip to mix the glycerol into solution, this took a while
  • Used the vacuum to sterile filter the solution through a 0.2um filter
  • Kept at room temp until the day of use

20240125 Overnight Culture

  • Set up 3 LB tubes with slightly less than 2mL in them
  • Added antibiotics:
    • 0.9ul stock Kanamycin
    • 0.55ul stock Chloramphenicol
  • Picked 3 colonies from the Colony 1 Chlor + Kan plate (see here, was still in fridge) and added to the LB tubes
  • Let bacteria grow overnight at 37C

20240126 Making Electrocompetent Bacteria

  • In the morning prepared a 500mL flask with 100mL of LB:
    • Mixed 250mL of DI water and 5g of Lennox LB with shaking
    • Aliquoitted 100mL to the 500mL flask
    • Aliquoited 2mL to a rack of test tubes
    • Autoclaved the LB on the short liquid cycle
    • Let the flask cool about 30 min before use on the bench and the tubes went in the 4C
  • After cooling, I added the antibiotics needed:
    • Kanamycin:
      • 100,000mg/mL * X = 20ug/mL * 100mL
      • X = 0.05mL or 50ul of stock Kanamycin
    • Chloramphenicol:
      • 50,000mg/mL * X = 20ug/mL * 100mL
      • X = 0.04mL or 40ul of stock Chloramphenicol
  • Mixed the flask with the antibiotics
  • Added 1mL of the overnight culture from one of the LB tubes to the flask
  • Placed the flask in the 37C incubator for 2.75 hours
  • During this time the 10% glycerol was placed in the 4C to cool down
  • After incubation (there was considerable growth/clouding of the media) the fluid was transferred to 2 50mL conicals and placed on ice
  • The following processes were done in the Egan Lab to use their centrifuge
  • Fast cooled their centrifuge while the bacteria was chilling on ice
  • Centrifuged the two 50mL conicals at 6,000rpm for 5 min at 4C
  • The supernatant was poured in a flask that would later be autoclave sterilized to pour the liquid down the sink
  • Added 40mL of cold 10% glycerol (kept on ice) and resuspended the pellets with a lot of vortexing
    • At this point I thought the spin was too strong because the pellets did not resuspend easily
  • Centrifuged 3 min 4C 4000 rpm
  • Poured off supernatant into waste, pellet kind of came off so I didn’t pour off everything
  • Added 40mL of cold 10% glycerol (kept on ice) and resuspended the pellets with a lot of vortexing
  • Centrifuged 3 min 6000rpm
  • Poured off supernatant into waste, pellet kind of came off so I didn’t pour off everything
  • Added 40mL of cold 10% glycerol (kept on ice) and resuspended the pellets with a lot of vortexing
  • Centrifuged 4 min 6000rpm
  • Poured off supernatant into waste, pellet kind of came off so I didn’t pour off everything
  • Added 40mL of cold 10% glycerol (kept on ice) and resuspended the pellets with a lot of vortexing
  • Centrifuged 7 min 6000rpm
  • Poured off everything in the supernatant into waste even though I lost some bacteria
  • Resuspended each pellet in 500ul of cold 10% glycerol
  • Aliquoited resuspention 50ul into 21 cryo tubes on ice
  • Aliquoited resuspention 100ul into 3 cyro tubes on ice
  • Froze cryotubes at -80 for storage until use later