Paulsson:Competent cells 070116

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Electrocompetent cell prep 01/16/07

  • Primary cultures grown overnight at 37deg in LB from single colonies of MC1061, PMB14, PMB27
  • Secondary cultures: measured in NanoDrop (NB: 1mm path length!!!)

MC1061: inoculated approx. (2x) 300ml LB with 1ml... 3.5hrs later OD600 ~ 0.06,
PMB14: inoculated approx. 300ml LB with 1ml... 3hrs later OD600 = 0.075
PMB27: inoculated approx. 300ml LB with 1ml... 3hrs later OD600 = 0.077

  • Followed CP method, using solutions and tubes pre-cooled in ice water baths, pipets and pipet tips pre-cooled at -20deg.

Washed with 275ml water.

Very loose pellet after water washes. Resuspended in approximately 45ml residual supernatant, then added 900ul 50% glycerol. Pelleted cells.
Aprrox. 450ul packed cells per tube. Resuspended with 500 - 600ul 10% glycerol in water, yielding approx. 1ml of cell suspension per 300ml culture. Dispensed in 95ul aliquots using cut tips (wide-mouth). Froze in pre-cooled (-20deg) microfuge tubes. Stored at -80deg. All labels have strain names in RED ink.

  • Transformation test

Thawed cells. Added 1ul 1ng/ul pPM3 pDNA. Flicked tube and transfered to 0.2cm e-poration cuvette. Zapped using MicroPulser on setting "Ec2". Immediately added 1ml SOC, mixed cells, transfered to culture tube. Shook at 30deg for 60 min.

Made serial 10-fold dilutions (1/10 to 1/10,000) and plated 100ul cells onto LB/Amp. Grew o/n at 30deg. (100 cells on 1/1000 dilution = 10(6) cells per ng of pDNA)

MC1061....... 6 x 10(9) cells / ug pDNA
PMB14........ 3 x 10(7) cells / ug pDNA
PMB27........ 3 x 10(7) cells / ug pDNA