One-step Transformation of E. coli

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].

3.For long-term storage,cells are frozen immediately in a dry ice/ethanol bath and stored at -70℃.

For transformation,a 0.1 ml aliquot of cells is pipetted into a cold polypropylene tube containing 1μl (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℃.

5.A 0.9 ml aliquot of TSS (or LB broth)plus 20 mM glucose is added,and cells are incubated at 37℃ 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.