PhiX:Transformation

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With credit to Art Poon for base protocol and Maniartis.

  • pre-chill centrifuge to 4oC and get ice
  • freeze two 20ml, two 10ml serological pipettes and 1ml, 200ul tip boxes - use these for all transfers
  • chill two 50ml centrifuge tubes
  • prepare 50ml LB + 1ml O/N C122 (or chosen strain; Art Poon says inoculate with colony)
  • follow until OD600nm = 0.250 (must catch in exponential phase)
  • using 20ml pipette, divide culture into two ~25ml aliquots into chilled centrifuge tubes
  • cool for 5 minutes on ice (Maniartis says 10 min)
  • centrifuge @ 2700g for 10 minutes at 4oC
  • discard supernatent - pellets should be pinky-nail size
  • using 10ml pipette, dispense 5ml ice-cold 0.1M CaCl2 to each tube
  • resuspend pellet
  • chill cells on ice for 30 mins
  • centrifuge @ 2700g for 10 minutes at 4oC - pellets should be ring-shaped
  • discard supernatent
  • using frosty 1ml tip, resuspend each pellet with 500ul ice-cold 0.1M CaCl2 (do not vortex)

At this point may store cells at +4oC for 24-48 hours: this increases efficiency.
For long-term storage: snap freeze in liquid nitrogen. Thaw for ten minutes on ice.

  • using frosty 200ul tip, transfer 100ul aliquots to (thin-walled) PCR tubes
  • add DNA (e.g., 5ul mutagenesis PCR or 1/3 ligation reaction) to each 100ul aliquot
  • chill on ice for 1 hour (meanwhile heat waterbath to 42oC)
  • heat shock: transfer tubes to waterbath, incubate for 1 minute exactly
  • return tubes to ice for a minute or two
  • optional: add cell/DNA mix to 500ul SOC in larger tube and incubate at 37oC for 45 minutes or fewer
  • spread plates with cell/DNA(/SOC) mixture and incubate O/N at 37oC

Alternatively if plating mutagenic phage:

  • transfer aliquots to soft agar with 100ul O/N C122 and MgCl2/CaCl2 solution for infection and plate
  • incubate plates for four hours at 37oC