tss-transformation

The Phage Cookbook by Dominik Refardt

TSS Transformation of E. coli with plasmids

TSS transformation is not super-efficient (as compared to transformation with electroporation), but it is much easier. As long as you just want to transform with plasmids, this is the method of choice. For applications that require a high efficiency (e.g. transformation with linear DNA), this method is not good enough. We successfully transformed pKD46, but were not able to transform pCP20 with this method.

Recipe for 2x TSS (Transformation and Storage Solution)

TSS is LB with 10% (wt/vol) polyethylene glycol (PEG), 5% (vol/vol) dimethyl sulfoxide (DMSO), and 50 mM Mg²⁺ (Chung et al. 1989). The recipe below is for a two-fold concentrated solution.

• 1. Dissolve in 50 mL ddH₂O: 0.8 g Bacto-Tryptone, 0.5 g Yeast Extract, 0.5 g NaCl, 20 g PEG 8000.

  2. Add 10 mL 1 M MgSO₄.

  3. Add 10 mL DMSO.

  4. Adjust pH to 6.5 (should already have that pH).

  5. Fill water to 100 mL.

  6. Filter-sterilize through a 0.2-µm filter. Don't even think about using a 10-mL syringe with an attached filter for this step. You need a big filter that fits on a bottle and can be attached to a vacuum pump.

  7. Store at 4° C or at -20 °C. The latter ensures that you will have no contamination growing in your TSS.

Transformation protocol

• 1. If you store TSS at -20 °C, take it out of the freezer and thaw it at room temperature, then place on ice.

  2. Inoculate target strain from a single colony in LB and grow till the culture is slightly turbid (OD₆₀₀ between 0.1 and 0.3).

  3. Chill on ice for 10 min.

  4. Add an equal volume of ice cold 2x TSS.

  5. Vortex thoroughly but avoid warming up the cells.

  6. Keep on ice for 30 min up to several hours (whatever is convenient). Even overnight storage works.

  7. The cells are now competent. Add 1 mL of cells to 1 µl plasmid DNA (should be at least 10 ng) in a pre-chilled 1.5-mL tube and vortex briefly.

  8. Incubate on ice for 30 min up to several hours (increasing incubation time enhances transformation efficiency).

  9. If selecting for ampicillin, plate immediately on selective plates, if selecting for any other resistance incubate at room temperature or 37 °C for 1 hour to allow for phenotypic expression.

  10. Before plating, cells can be concentrated by a 1 min spin in a benchtop centrifuge. Remove most supernatant and resuspend in the leftover by vortexing.