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Toxoplasma gondii is a kind of obligate intracellular parasites that are capable of infecting virtually all warm-blooded animals. It is one of the most common parasites in human. Serological studies estimated that up to a third of the global population has been chronically infected with the parsites. Toxoplasma gondii is also used as a model orgnisim of Apicomplexans that includes Plasmodium – the parasites that cause malaria. Gene knockout is a very important way to study gene function in all organisms. When it comes to Toxoplasma, it’s very difficult as this haploid parasite has strong adaptability to circumvent the condition of gene defects by gene duplication. High throughput screening at very early stage of transfection is very important for generating a true knockout of this parasite. For some genes that are required forToxoplasma gondii, the high throughput screening is necessary. Different labs use different protocols. After reading and practicing protocols from different papers or different labs, here I established an efficient pipeline for this purpose.