Preparation of Chemically Competent Cells

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

Materials

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

Protocol

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.