Procedure:

1. Have nitrocellulose membrane ready, draw grid by pencil to indicate the region which are going to blot.
2. Using a narrow-mouth pipette tip, spot 2 µL of sample onto the nitrocellulose membrane at the center of the grid. Minimize the area that the solution penetrates (usually 2–4 mm diameter) by applying it slowly.
   
3. Let the membrane dry.
   
4. Block non-specific sites by soaking in 5% BSA in TBS-T in a 10 cm Petri dish (30 min to 1 h at room temperature.
   
5. Incubate with primary antibody (0.1–10 µg/mL for purified antibody, 1:1,000–1:100,000 for antisera, 1:100–1:10,000 for hybridoma supernatant) in BSA/TBS-T for 30 min at room temperature.
   
6. Wash three times with TBS-T (3 x 5 min).
   
7. Add 1 ml G/L-M17B + 20 mM MgCl₂ + 2 mM CaCl₂.
8. Keep the cuvette for 5 min on ice and incubate 1-1.5 h at 30 °C.
9. Plate 10 µl, 100 µl, 900 µl on M17agar with glucose or lactose and antibiotics (depends on plasmid) - Incubate 1-2 days at 30 °C.

Tips:

1. Lactococcus lactis grows very slowly in G/L-SGM17B. Leaving out the sucrose is possible but can lower the transformation efficiency. The medium for cell recovery must contain MgCl₂ and CaCl₂.
2. The sterilization temperature can be set at 108 degrees Celsius in case of carbonization.

Applications in our project: Transformation of BBa_K2309005, BBa_K2309004 and BBa_K2309028 (independent in pNZ8148)