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TRANSFORMATION (ONE-STEP PEG METHOD)

TRANSFORMATION (ONE-STEP PEG METHOD)

  1. Dilute 1:100 a fresh O/N culture of bacteria into prewarmed LB broth and incubate cells with shaking (225 rpm) to an OD600 of 0.3-0.4.
  2. Add an equal volume of ice-cold 2X TSS and mix gently. [TSS is LB broth with 10% PEG (MW3350-8000), 5% DMSO, and 20-50 mM Mg2+ (MgSO4 or MgCl2) at a final pH of 6.5].
    • For long-term storage, cells are frozen immediately in a dry ice/ethanol bath and stored at -70 C.
    • For transformation, a 0.1 ml aliquot of cells is pipetted into a cold polypropylene tube containing 1 ul (100 pg) od plasmid DNA, and cell/DNA suspension is mixed gently. [When frozen cells are used, cells are thawed slowly on ice and used immediately.]
  4. The cell/DNA mixture is incubated for 5-60 min at 4 C.
  5. A 0.9 ml aliquot of TSS (or LB broth) plus 20 mM glucose is added, and cells are incubated at 37 C with shaking (225 rpm) for 1 hour to allow expression of the antibiotic-resistance gene.
  6. Transformants are selected by standard methods.

PNAS (1989) 86:2172-2175. CT Chung, SL Niemela, RH Miller.