Irdye 800cw streptavidin conjugated dye

Genomic DNA was isolated from parasites using the QIAamp DNA blood kit (Qiagen). Control primers to amplify the genome were P4 and P12, and primers used to amplify integrated DNA were P12 and P13.
Southern blot analysis was performed on DNA isolated from PfGRP170‐GFP‐DDD parasites (1B2 and 1B11) as described previously (Cobb et al., 2017; Florentin et al., 2017). The assay was also performed on PM1KO parental DNA and the pGRP170‐DDD plasmid as a control. DNA was isolated from parasites using the QIAamp DNA blood kit (Qiagen). A total of 10 μg of precipitated PM1KO DNA, 1B2, and 1B11 DNA and 10 ng of pGRP170‐DDD plasmid were digested overnight with Mfe1 (New England Biolabs). The biotinylated probe used was generated by PCR using biotinylated‐16‐UTP (Sigma) and primers P3 and P4. The biotinylated probe on the southern blot was detected using IRDye 800CW streptavidin‐conjugated dye (LICOR Biosciences) and imaged using the Odyssey infrared imaging system (LICOR Biosciences).

Southern blotting was performed with genomic DNA isolated using the Qiagen Blood and Cell Culture kit. Ten micrograms of DNA was digested overnight with NcoI/XmnI for PfHsp70x-DDD and BamHI/ScaI for PfHsp70x-KO (New England Biolabs). Integrants were screened using biotin-labeled probes against the 3′ end (PfHsp70x-DDD parasites) or 5′ end (PfHsp70x-KO parasites) of the pfhsp70x open reading frame (ORF). Southern blotting was performed as described earlier (43 (link)). The probe was labeled using biotinylated biotin-16-dUTP (Sigma). The biotinylated probe was detected on blots using IRDye 800CW streptavidin-conjugated dye (LICOR Biosciences) and imaged, processed, and analyzed using the Odyssey infrared imaging system software (LICOR Biosciences).