To begin, verify the sequence of the desired genes isolated from the drug-treated Plasmodium falciparum parasites. After diluting the parasitized RBCs, add an appropriate volume to achieve a total of 50 parasites in 10 milliliters of media, ensuring 2%hematocrit. Then, add 100 microliters of this media containing 50 parasites to each well of the 96-well plate.
Place the plate in an airtight Secador flushed with mixed gas. And incubate at 37 degrees Celsius, replenishing the media every two days. Replace the media with complete RPMI containing 2%hematocrit every seventh day until the parasites appear.
Tilt the 96-well plate to a 45-degree angle to let the red blood cells settle down. Carefully remove the media from each well using a multi-channel pipette. Observe a color change to yellow or dark red in wells containing parasites, which contrasts with the pink color of media in parasite-free wells.
Next, take out a small amount of culture from the well exhibiting the color change and prepare a smear on a labeled slide. After performing Giemsa staining, observe the slide under a microscope. Transfer the contents of the well where parasites were observed into a T25 flask and incubate to let the parasites grow for four to five days.
Once the parasitemia reaches two to 3%saponin lyse 500 microliters of parasite culture. And extract the parasite genetic material.
