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 previously32 (link),35 (link). 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). 10μg of precipitated PM1KO DNA, 1B2, and 1B11 DNA and 10ng of pGRP170-DDD plasmid was 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 blot analysis was performed on DNA isolated from PfGRP170-GFP-DDD parasites (1B2 and 1B11) as described previously32 (link),35 (link). 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). 10μg of precipitated PM1KO DNA, 1B2, and 1B11 DNA and 10ng of pGRP170-DDD plasmid was 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).