Paulsson:Competent cells 061127

From OpenWetWare
Jump to navigationJump to search

Home        Group Meeting        Protocols        Inventory        Journal Watch        Top 100        Links        Facts of Life       

Electrocompetent cell prep 11/27/06

  • Primary cultures grown overnight at 37deg in LB from single colonies of MC1061 and DH5-alpha

DH5-alpha: 1/10 dilution, OD600 = 0.355, measured in BioRad spectrophotomer in Kirschner Lab
MC1061: 1/10 dilution, OD600 = 0.385

  • Secondary cultures:

DH5-alpha: inoculated approx. 550ml LB with 2.5ml... 3.5hrs later OD600 = 0.507
MC1061: inoculated approx. 550ml LB with 2.3ml... 3.5hrs later OD600 = 0.553

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

Washed with 350ml water instead of 500ml (pooled cells from 2x 500ml tubes into one).

Very loose pellet after water washes. Resuspended in approximately 35ml residual supernatant, then added 45ml 10% glycerol. Transfered to two 50ml conical tubes. Pelleted cells.
Aprrox. 200ul packed cells per tube. Resuspended with 400ul total, yielding approx. 800ul of cell suspension. Dispensed in 50ul aliquots using cut tips (wide-mouth). Froze in pre-cooled (-20deg) microfuge tubes. Stored at -80deg.

  • Transformation test

Thawed cells. Added 1ul 1ng/ul ts-Rep 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 40min.

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)

note, strains were treated nearly identically, but transformation efficiency was dramatically different:
MC1061....... 1.5 x 10(9) cells / ug pDNA
DH5-alpha... 5.1 x 10(7) cells / ug pDNA