TSS Buffer | 100ml | 20 Preparations

TSS Buffer- GMES modified recipe

Transformation and Storage Solution (TSS), containing polyethene glycol, dimethylsulfoxide and divalent cations in a growth medium, is used for the one-step preparation of competent E. coli cells and the transformation of E. coli strains, even without heat shock.

TSS is an alternative way to the chemical transformation method using CaCl₂. 0.22μm filtration plus 5min 275/365nm UV treatment sterilized. Can be stored at room temperature. Suggest storage at 4°C / 39.2°F  refrigerator.

Advantage:
One-step competent cell preparation/transformation, only fresh early log phase E. coli need to be suspended in TSS solution.

Disadvantages:
Relatively lower transformation efficiency than the CaCl₂ method.

Notes:

- Use only for the transformation of plasmids, not for ligation mixtures in molecular cloning.

- It is possible to transform at least two plasmids at the same time.

- CaCl₂ transformation does not work for E. coli strains W3110 and W3110 Z1, so TSS transformation is the method of choice.

The formulation of this buffer solution has been modified by GMES to maximise the transformation rate.

Components in 100ml

Competent cells Preparation

Step 1. Inoculate 1mL of SOC medium with E. coli strain of choice in a 1.5mL PP tube (snap-cap) and culture overnight at 37°C with rotation.

Step 2. Prepare 100mL of liquid medium LB in a sterile 250mL Erlenmeyer flask. DNA booster can be applied with the LB to harvest more and robust cells.

Step 3. Seed the 1mL culture into the 250mL flask and incubate at 37°C with 230 rpm rotation until the OD600 reach 0.3 to 0.4 (0.3 ≤ OD600 ≤ 0.4) ( approximately 2-4h).

Step 4. Once the proper optical density has been achieved, transfer the 100mL of culture into 2x 50mL PP centrifuge tubes and centrifuge under 3,000g for 10 min at 4°C. Concentrate the cells by discarding the supernatant.

Step 5. Resuspend each tube in 5mL of ice-chilled TSS buffer with gentle vortexing.

Step 6. Chill TSS-suspended cells on ice for 15 min. Prepare/label/pre-chill 50x 1.5mL PP tubes (snap-cap) during this time.

Step 7. Aliquot 100~200 μL of TSS-suspended cells to each 1.5mL PP tube while ensuring the cells remain well mixed.

Step 8. Competent Cells can be used immediately or stored at -80°C.

Rapid transformation without heat shock

Step 1. Inoculate 3-5 ml of LB medium with colonies from a fresh agar plate. DNA booster can be used with LB to increase the number of cells and save incubation time.

Step 2. Incubate at 37 °C and 230 rpm for 1.5 to 2.0 hours (exponential growth, OD600 0.2-0.5). The culture should become turbid).

Step 3. Centrifuge at 5000xg for 5 minutes at 4°C and concentrate the cells by discarding the supernatant.

Step 4. Resuspend each tube in 100µl of ice-chilled TSS buffer with gentle vortexing.

Step 5. Prepare 1.5 ml tubes with 200 µl of TSS buffer and keep ice-chilled. 

Step 6. Add 50µl cells and 0.5 - 1.0 µl Plasmid. Vortex and incubate on ice for 20 - 30 min. Transformation efficiency decreases with longer incubation times!

Step 7. Incubate for 45~60 min at 37 °C on the Thermomixer with shaking (900 rpm) or incubate at 37 °C and 350 rpm. 

Step 8. Spread the incubated fluid onto agar plates containing the appropriate antibiotic.