Journal: BMC Biology

Article Title: Involvement of Plasmodium falciparum protein kinase CK2 in the chromatin assembly pathway

doi: 10.1186/1741-7007-10-5

Figure Lengend Snippet: Reverse genetics of PfCK2β1 . 3D7 parasites transfected with pCAM-BSD-KOPfCK2β1 with or without pCHD-PfCK2β1 were analyzed by PCR and Southern blotting. (A) Locations of the primers used for PCR screening, and the restriction enzymes used to cut the genomic DNA to give a diagnostic pattern of bands for analysis by Southern blotting. One truncated copy possesses its promoter and initiation codon, but lacks the C-terminal cysteine pair, a stop codon and a 3'UTR, whereas the other copy possesses both cysteine pairs but lacks a promoter and an initiation codon, and has an artificial stop codon introduced. (B) PCR screening of DNA from untransfected 3D7 parasites, two separate pCAM-BSD-KOPfCK2β1-transfected lines (KOCK2β1 1 and KOCK2β1 2), and parasites transfected with both the knockout plasmid and the complementation plasmid (KOCK2β1 + comp). (1) Amplification of the wild-type locus; (2) amplification over the 5' integration boundary; (3) amplification of the insert in the pCAM-BSD-KOPfCK2β1 plasmid. Evidence of integration was seen only in the DNA from the doubly transfected parasite culture (KOCK2β1 + comp; lane 2, faint band seen at 598 bp). (C) The parasite DNA was digested using the restriction enzymes EcoR I and Cla I, and analyzed by Southern blotting, using BSD and PfCK2β1 sequences as probes. (1) Untransfected 3D7; (2) KOCK2β1 1; (3) KOCK2β1 2; (4) KOCK2β1 + complement. See text for details.

Article Snippet: Forty-eight hours later, the ring-stage parasites were transfected by electroporation with 100 μg of purified plasmid DNA in buffer (Cytomix; Gibco-BRL, Grand Island, New York, NY, USA) as described previously [ ].

Techniques: Transfection, Southern Blot, Diagnostic Assay, Knock-Out, Plasmid Preparation, Amplification