I am attempting to prepare a glycerol stock for Stbl3 cells from commercially competent cells for Stbl3. I followed these procedures to prepare the glycerol stock, and I would greatly appreciate it if someone could review and confirm whether my method is correct.

Step 1: Mixed 45 µL of Stbl3 competent cells with 300 µL of LB broth (no antibiotics). Step 2: Shook the mixture for 1 hour at 37°C. Step 3: Plated 200 µL of the mixture on an LB agar plate (without antibiotics, as there is no plasmid to select for). Step 4: Spread the mixture across the plate using a hockey stick. However, after incubation, the plate was fully covered with bacterial growth, with no specific or isolated colonies (as seen in the attached image). I decided to use a sterile loop to pick a single portion of the growth and inoculate it into 5 mL of LB broth (without antibiotics). I placed the culture in the shaker for overnight incubation. then the OD600 is 2.218, then I mixed 500 µL of this bacterial culture with 500 µL of 50% glycerol to create the glycerol stock.

then incubated at room temperature for 20 minutes then stored at -80

Question:

1- Is this procedure correct, or do you suggest plating the 45 µL of Stbl3 cells directly onto an LB agar plate (without recovery in LB broth)?

2- Would it be better to create streaks for single colonies on the plate rather than spreading with a hockey stick?

thank you in advance for your assistance