(A) Long range PCR analysis of genomic DNA from WT, PfΔslarp and PfΔb9Δslarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B) and PCR products are also digested with restriction enzymes x (XmaI) and kx (KpnI/XcmI) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δslarp-a, 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δslarp-b, 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δb9-b, 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). (B) Southern analysis of restricted genomic DNA from WT, PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7, and PfΔb9Δslarp-G9 asexual parasites. DNA was digested with restriction enzyme (E: TaqI) and probed with the 5′ slarp targeting region (P: 5′ slarp-T; see A) on the left side of the slarp Southern or probed with the 3′slarp targeting region (P: 3′ slarp-T; see A) on the right side of the slarp panel. For analysis of the b9, integration DNA was digested with restriction enzymes (E: RcaI) and probed with the 5′ b9 targeting region (P: 5′ b9-T; see A) on the right panel. The expected fragment sizes are indicated in panel (A). (C) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔslarp-a, PfΔslarp-b, PfΔb9Δslarp-F7, and PfΔb9Δslarp-G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B) and generated the expected 130 bp fragment.