To begin, add 200 microliters of 100 millimolar sodium chloride to a tube containing transfected trophozoites of naegleria gruberi. Centrifuge the tube at 600G for 10 minutes at 4 degrees Celsius. After removing the supernatant, resuspend the cell pellet in 100 microliters of 100 millimolar EDTA.
Place the tube in a heat block for 15 minutes at 98 degrees Celsius to lyse the cells. Centrifuge the tube at 4 degrees Celsius for 10 minutes at top speed to pellet the debris. Transfer the supernatant into a new tube.
Next, add 0.5 volumes of ammonium acetate, three volumes of absolute ethanol, and one microliter of glycogen to the supernatant. Invert the tube several times to mix, before incubating it at minus 80 degrees Celsius for 30 minutes or overnight. After incubation, centrifuge the tubes at room temperature for 10 minutes at 16, 000G.
Remove the supernatant without disturbing the pellet, and add 200 microliters of 70%ethanol to wash it before centrifuging. Resuspend the dried pellet in sterile deionized water to achieve a trophozoite density of 10, 000 trophozoites per microliter. After PCR with primer specific for the transfected DNA and detection of the transformed trophozoites, add two microliters of 6X loading dye to the CERE samples.
Then, load the dyed samples onto a 0.8%agarose gel. After electrofluorescing the gel for 1.5 to 2 hours, incubate it in DNA dye diluted in 100 milliliters of deionized water. Transfer the stained gel into deionized water for 20 minutes to destain it.
Visualize the gel on an ultraviolet light system to document the results. PCR analysis showed that pGRUB was detected in transfected trophozoites through at least seven passages. pGEM was lost after the first passage, indicating that the empty plasmid was not retained by the amoebas.
