Editing Competent-Cells (section)

== TSS Method ==

TSS Buffer

5g PEG 8000<br/>1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H20)<br/>2.5 mL DMSO<br/>Add LB to 50 mL<br/>Filter sterilize (0.22 μm filter)

Grow a 5 mL seed culture of cells in LB medium to saturation.&nbsp;

Dilute this culture back into 25–50 mL fresh LB in a 200 mL conical flask (by at least 1/100.by at least 1/100.)

Grow the diluted culture to an OD600 = 0.2–0.5.

Put eppendorf tubes on ice now so that they are cold when cells are aliquotted into them later.&nbsp;

Split the culture into two 50 mL falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at 4°C and the cells should be kept on ice wherever possible

Centrifuge for 10 min at 3000 rpm and 4°C.

Remove supernatant. The cell pellets should be sufficiently solid that&nbsp; the supernatant&nbsp; can be just poured off.

Pipette out any remaining media.

Resuspend cells in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume.&nbsp;

Vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.

Higher concentrations of cells (2–3×) are found to enhance efficiency, requiring cell resuspension in 3–5% culture volume of TSS instead of 10% (Dueber & Bennett Lab).

Add 100 µL aliquots to chilled eppendorfs and store at -80°C.