Preparing chemically competent cells

Preparation

1. Grow a 5 - 25 mL culture to an OD₆₀₀ of 0.2 - 0.5.
2. Centrifuge for 10 minutes at 3000 rpm and [math]\displaystyle{ 4^o }[/math]C.
3. Remove supernatant and replace with 10% original volume TSS.
4. Make 100 uL aliquots and store at [math]\displaystyle{ -80^o }[/math]C.

Use

1. Thaw TSS cells on ice.
2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
   • Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
3. Let sit for 30 minutes on ice.
   • Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
4. Incubate cells for 30 seconds at [math]\displaystyle{ 42^o }[/math]C.
   • Note: According to the original TSS paper and qualitative experience, this step is completely optional and may actually reduce transformation efficiency.
5. Add 1 mL SOC (2XYT and LB are also suitable) at room temp.
6. Incubate for 1 hour at [math]\displaystyle{ 37^o }[/math]C.
   • Note: Can also save some time here by reducing incubation to ~45 min.
7. Plate 200 µL onto plate with appropriate antibiotic.

Buffers

TSS (50 mL)

• 5g PEG 8000
• 1.5 mL 1M MgCl2
• 2.5 mL DMSO
• LB to 50 mL

Filter sterilize (0.22 µm filter) and store at 4 ˚C or -20 ˚C.