Reagent: TSS (Transformation and Storage Solution for chemical transformation)

• 85 % LB medium
• 10 % PEG (wt/vol, MW 8000)
• 5 % DMSO (vol/vol )
• 50 mM MgCl2 (pH 6.5)

1. Streak the cell stock on a LB plate (added antibiotic if cells have antibiotic resistant). Incubate the plate at 37^oC overnight.

2. Pick a single, well-isolated colony and inoculate it into 5 ml of LB broth (plus antibiotic). Incubate at 37 ^oC overnight with shaking at 220 rpm.

3. Transfer 1 ml of the saturated overnight culture to a sterile 500-ml flask containing 100 ml of LB medium (do not add antibiotic at this step). Incubate the cells at 37 ^oC with the shaking at 220 rpm, until OD600 reach 0.5 ; this usually takes 2.0-2.5 hr. Check the OD frequently when it gets beyond 0.2 to avoid overgrowth.

4. When the culture reaches an OD600 of 0.5, chill the flask on the ice for 20 min and then collect the cells by centrifugation at 1500 rpm for 5 min at 4 ^oC.

5. Resuspend the cells in 10 ml of ice-cold TSS solution. Now the competent cells are ready to be transformed.

6. Aliquot 150 ul competent cells to 1.5ml tube. If they are not immediately used, cells can be stored at 4 ^oC for maximum of 6 hr without significant loss of competency. The same competent cells can also be stored at -70 ^oC for long-term storage (pre-treat with liquid nitrogen or dry ice).

(p.s.) Competent cells should give a minimum of 1x10⁶ transformants per ug of plasmid DNA.

Transformation frequency of frozen cells is 30 % of that of the fresh cells, when used within two months.

Transforming the cells.
1.  Add DNA (< = 20  ul ) to ice cold 150 ul competent cells.  Thaw -70 ^oC competent cells first by hand temperature.

You can transform smaller portions of cells (eg. 75 ul or 50 ul), but you should decrease the volumes of DNA and media proportionately.

3.  Heat shock at 42 ^oC for 2 min.

4.  After heat shock, put on ice for 2 min.

5.  Add 0.8 ml LB broth.

Many transformation protocols indicate that you should allow cells to recover in SOC (2% Bactotryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM glucose).  I think this improves the number of colonies you get.

7.  Plate (< 200 ul ) on the appropriate agar plates which contain antibiotic.

8. Incubate plates at 37 ^oC, overnight.

Reference
Chung, C. T. et al. (1989) One-step preparation of competent E. coli: Transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86:2172-2175.