Difference between revisions of "Team:XJTLU-CHINA/Protocols Electroporation"
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Latest revision as of 08:35, 31 October 2017
Electroporation for L. lactis
Materials:
Procedure:
- Place 40 μl competent cells in a pre-chilled electroporation cuvette with 1 μl DNA (reconstituted in TE-buffer) and keep the cuvette on ice.
- Use the Electroporator with following adjustments:
2500 V (12.5 KV/cm)
25 μF
200 Ω - Pulse (normal reading is 4.5-5 msec) - Add 1 ml G/L-M17B + 20 mM MgCl2 + 2 mM CaCl2.
- Keep the cuvette for 5 min on ice and incubate 1-1.5 h at 30 °C.
- 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:
- 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.
- 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)
Collaborators and Supporters
