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Preparation of Chemically Competent Cells

This protocol involves steps occurring over multiple days. Prepare accordingly.


  • TSS Buffer (see recipe below)
  • LB broth
  • Bacterial stock (ex. DH5-α)
  • 1.5 mL microcentrifuge tubes


Day 1

  1. Streak bacteria from a glycerol stock on an LB agar plate and incubate the plate at 37°C overnight.

Day 2

  1. Inoculate a 5 mL LB broth culture (no antibiotic) using a single colony from the plate in step 1. Incubate the culture overnight at 37°C.

  2. Prepare TSS buffer using the following recipe:

Reagent Amount
PEG 8000 (or 3350) 5 g
MgCl2, 1M 1 1.5 mL
DMSO 2.5 mL
LB Broth up to 50 mL

1 0.3 g MgCl2*H2O can be used in place of the 1M MgCl2 solution.

  1. Filter the TSS buffer through a 0.22 μm filter and store at 4°C.

Day 3

  1. Dilute the culture 1:100 by inoculating four 50 mL conical tubes with 50 mL LB broth and 500 μL of overnight bacterial culture. Grow the cultures at 37°C until the OD600 is between 0.2 and 0.5.

  2. Chill the culture, 1.5 mL microcentrifuge tubes and TSS on ice 10 min. Meanwhile, cool the centrifuge to 4°C for the next step.

  3. Pellet the bacterial cells at >3000 RCF for 10 min at 4°C.

Important: The cells must remain on ice and stay cold from this point forward in order to maintain a high transformation efficiency.

  1. Remove the supernatant and resuspend in 10% volume chilled TSS buffer (ex. resuspend a 50 mL culture in 5 mL TSS buffer)

  2. Aliquot 100 μL cells in TSS buffer into the chilled 1.5 mL microcentrifuge tubes.

  3. Flash freeze the aliquots in liquid N2 and store at -80°C.

The transformation efficiency should be tested for every new batch of chemically competent cells.