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
- Streak bacteria from a glycerol stock on an LB agar plate and incubate the plate at 37°C overnight.
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.
Prepare TSS buffer using the following recipe:
|PEG 8000 (or 3350)||5 g|
|MgCl2, 1M 1||1.5 mL|
|LB Broth||up to 50 mL|
1 0.3 g MgCl2*H2O can be used in place of the 1M MgCl2 solution.
- Filter the TSS buffer through a 0.22 μm filter and store at 4°C.
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.
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.
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.
Remove the supernatant and resuspend in 10% volume chilled TSS buffer (ex. resuspend a 50 mL culture in 5 mL TSS buffer)
Aliquot 100 μL cells in TSS buffer into the chilled 1.5 mL microcentrifuge tubes.
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.