This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache


  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.]
  3. The cell/DNA mixture is incubated for 5-60 min at 4 C.
  4. 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.
  5. Transformants are selected by standard methods.

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