TSS Competent Cells (For KCM Transformations)

TSS Competent Cells (for KCM Transformations)

Day 1
Restreak the bacteria on LB plates with appropriate antibiotics from a -80 stock.

It is best not to restreak from a competent cell stock.

Day 2

1. Grow up a single colony in 10mL of LB+antibiotic.
2. Autoclave large centrifuge bottles and hundreds of eppendorf tubes in covered beaker(s).
3. Prepare 1L LB in a flask.
4. Prepare the TSS solution:

Autoclave individually:

85mL LB
10g PEG-3350 in 5mL H2O (other PEG sizes are ok)
5mL DMSO
2mL 1M MgCl2

Allow those to cool, then combine and fridge.

Day 3

1. Prechill:

TSS solution (autoclaved and mixed)

Centrifuge rotor (well-cleaned)

Centrifuge bottles (autoclaved)

Beaker(s) of eppendorf tubes (autoclaved)

25-50mL serological pipette (sterile)

5mL repeating pipette tip (sterile)
2. Add 10mL overnight culture to pre-warmed 1L LB.
3. Grow culture to OD=0.5 (takes between 2-3 hours).
4. Shake on ice to stop the growth.
5. Spin cells at 3500rpm for 15min or 6500 for 5 minutes.
6. Resuspend the pellets in the ICE-COLD TSS using serological pipette.
7. Aliquot 200uL into eppendorf tubes using repeating pipette in the cold room.
8. Freeze cells in liquid nitrogen bath and store in -80.