Mahoneykurpe: Created page with "Based on protocol for ''E. limosum'' described in Sanford & Woolston (2022) [1], with some adjustments made based on results from initial electroporation tests which gave a transformation efficiency of approx. 6,000 CFU/µg in a best attempt. === '''Electrocompetent cell preparation''' === # Grow six 10 ml SA11 cultures (or upscale as required) to early log phase (OD ~0.5) # Cool cultures on ice for 20 min. # Bring cultures into chamber on ice, pour 30 ml culture into..."

2026-10-30T08:00:20Z

Created page with "Based on protocol for ''E. limosum'' described in Sanford & Woolston (2022) [1], with some adjustments made based on results from initial electroporation tests which gave a transformation efficiency of approx. 6,000 CFU/µg in a best attempt. === '''Electrocompetent cell preparation''' === # Grow six 10 ml SA11 cultures (or upscale as required) to early log phase (OD ~0.5) # Cool cultures on ice for 20 min. # Bring cultures into chamber on ice, pour 30 ml culture into..."

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Based on protocol for ''E. limosum'' described in Sanford & Woolston (2022) [1], with some adjustments made based on results from initial electroporation tests which gave a transformation efficiency of approx. 6,000 CFU/µg in a best attempt.

=== '''Electrocompetent cell preparation''' ===

# Grow six 10 ml SA11 cultures (or upscale as required) to early log phase (OD ~0.5)
# Cool cultures on ice for 20 min.
# Bring cultures into chamber on ice, pour 30 ml culture into two O-ring sealed Oak Ridge centrifuge tubes, ensure lids are sealed tightly and remove from anaerobic chamber, keeping on ice.
# Centrifuge at 15,000 × ''g'' at 4 °C for 10 min.
# Bring tubes back into chamber and pour off culture supernatant and add 30 ml ice cold 270 mM sucrose buffer, gently resuspending with a ≥5 ml pipette tip.
# Repeat buffer wash and centrifugation steps under the same conditions, but also including 20% (v/v) glycerol in the wash buffer.
# Bring washed pellets back into chamber, decant supernatant and remove as much excess buffer as possible without disturbing pellet.
# Add 300 µl 270 mM sucrose/20% glycerol buffer and gently resuspend as minimally as needed.
# Dispense preferred volumes into chilled Kimax tubes ensuring lids are securely fastened before removing from chamber.
# Store aliquots at -80 °C for later use.

=== '''Electroporation''' ===

# Thaw electrocompetent cell aliquots on ice, bring into anaerobic chamber with pre-chilled 2 mm electroporation cuvettes, CO<sub>2</sub> -filled Kimax tubes for recoveries, anaerobic M2GSC recovery medium, and pJIR750 plasmid DNA.
# Add 50 µl electrocompetent cells and 100-200 ng of pJIR750 plasmid to an electroporation cuvette, flick to mix and chill on ice for 3 min.
# Electroporate using the following settings: exponential decay, 2.5 kV, 400 Ω, 25 μF.
# Pipette 950 µl of M2GSC medium into the cuvette, mix gently and pipette into Kimax tube.
# Recover at 39 °C for six hours before plating 100 µl on M2GSC agar plates with 5 µg/ml thiamphenicol and incubate at 39 °C. Colonies usually form on the second day of incubation.

== External Links ==
Sanford PA, Woolston BM. 2022. Expanding the genetic engineering toolbox for the metabolically flexible acetogen ''Eubacterium limosum''. J Ind Microbiol Biotechnol 49: kuac019.

Eubacterium maltosivorans SA11 transformation protocol (BSI) - Revision history