Paulsson:Competent cells 061219

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Electrocompetent cell prep 12/19/06

  • Primary cultures grown overnight at 37deg in LB from single colonies of XL1-Blue and TOP10

XL1-B: 1/10 dilution, OD600 = 0.042, measured on Nanodrop in triplicate
TOP10: 1/10 dilution, OD600 = 0.035

  • Secondary cultures:

XL1-B: inoculated approx. 600ml LB with 2.5ml... 3.25hrs later OD600 = 0.055
TOP10: inoculated approx. 600ml LB with 2.6ml... 3.25hrs later OD600 = 0.079

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

Split culture into two 500ml centrifuge tubes.
Washed each with 350ml water.
Resuspended cells in residual supernatant (~30ml). Pooled cells and brought volume to ~400ml with water.
Very loose pellet after water washes. Resuspended in ~30-40ml residual supernatant, then added 10% glycerol to ~100ml. Transfered to two 50ml conical tubes. Pelleted cells.
for XL1-Blue: ~250ul packed cells per tube. Resuspended to ~1ml of cell suspension.
for TOP10: ~400ul packed cells and more sup. Resuspended to ~1.6ml total pooled.
Dispensed 50ul or 100ul aliquots using cut tips (wide-mouth) to pre-cooled tubes on bed of dry ice. Stored at -80deg.

  • Transformation test

Thawed cells. Added 1ul 1ng/ul pST11 pDNA. Flicked tube and transfered to 0.2cm e-poration cuvette. Zapped using MicroPulser on setting "Ec2". Immediately added 950ul SOC, mixed cells, transfered to culture tube. Shook at 30deg for 75min.

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

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