Electroporation of T. cruzi epimastigotes

Gather all necessary materials including 4 mm electroporation cuvettes, linear DNA donor, single guide RNA Cas9 plasmid DNA, and Trypanosoma cruzi epimastigotes suspended in cold cytomix buffer. All work is performed under sterile conditions in a biosafety cabinet.

Add both DNA suspensions to the internal wall of a sterile 4 mm cuvette, then add 400 microliters of cell suspension. Close the cuvette and incubate on ice for 10 minutes to allow mixing.

Turn on the electroporator and select the exponential protocol with settings of 1500 volts, 25 microfarads capacitance, infinite resistance, and 4 mm cuvette size.

Remove the cuvette from ice, wipe its external metallic sides, and insert it into the chamber with proper orientation. Apply three electric pulses by pressing the red button, ensuring the time constant displays between 0.8 and 1 millisecond, leaving 1 minute on ice between pulses.

After applying all three pulses, leave the cells on ice for 1 minute, then incubate at room temperature for approximately 15 minutes to allow recovery.

Return to the biosafety cabinet and transfer the electroporated cells into a sterile T25 flask containing 5 ml of culture medium supplemented with 20% fetal bovine serum.

Place the flask in an incubator at 28 degrees Celsius for 24 hours, after which selective antibiotics will be added to the culture.