TSS Buffer Composition

Protocol

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

2. Prepare 100mL of liquid medium (LB or SOC) in a sterile 250mL Erlenmeyer flask.

3. Seed the 1mL culture into the 250mL flask and incubate at 37°C with 225 rpm rotation.

-Use this time to prepare the TSS buffer

4. Cultivate until the optical density of the culture under 600nm wavelength is 0.3 to 0.4 (0.3 ≤ OD₆₀₀ ≤ 0.4) (should take approximately 2-3h).

-TSS buffer should be chilled by this point.

5. Once the proper optical density has been achieved, transfer the 100mL of culture into 2x 50mL PP centrifuge tubes and centrifuge under 2,700xg for 10 min at 4°C.

6. Resuspend each tube in 5mL of pre-chilled TSS buffer with gentle vortexing (≤50%).

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

8. Distribute 200 μL of TSS suspended cells to each 1.5mL PP tube while ensuring the cells remain well mixed.

NOTE: 100 μL will be used per transformation.

9. Cells can be used immediately, or stored at -80°C.