Cdp star chemiluminescence substrate

Cultures synchronized to 5–8% ring-stage parasitaemia after sorbitol treatment were used for DNA transfections. Plasmids were transfected into parasites suspended in Cytomix solution (120 mM KCl, 150 µM CaCl₂, 10 mM K₂HPO₄/KH₂PO₄, 25 mM HEPES, 2 mM EGTA, 5 mM MgCl₂ pH 7.6) (Sigma-Aldrich) by electroporation in a Gene Pulser Xcell (0.31 kV, 950 µF) (Bio-Rad). After electroporation, parasites were resuspended in 2 ml of complete RPMI and transferred to a T25 culture flask containing 8 ml of complete RPMI-1640, adjusted to 4% haematocrit, gassed and incubated at 37 °C. Approximately 2 h post transfection, media was replaced with 9.5 ml of fresh complete RPMI and drug selection was applied for the resistance marker in the transfected plasmid the following day.
For the Southern blot in Fig. 6c, 2.5 μg of parasite genomic DNA and 1 ng of pCAM-Pfnek1 plasmid DNA were digested with EcoRV, separated on a 1% agarose gel and transferred to a nylon membrane. The blot was probed with the digoxigenin (DIG)-labelled Pfnek1 sequence amplified from 3D7 genomic DNA with the primer pair 97J/98J (used to produce the pCAM-Pfnek1 truncation construct) and incubated with an anti-DIG antibody conjugated to alkaline phosphatase (Roche). Chemoluminescence detection was performed using CDP-star Chemiluminescence substrate (Roche) and imaged using a Bio-Rad ChemiDoc XRS + imaging system.

For detection with strand-specific probes the nts 16 177–40 of human mtDNA were cloned into the pCR2.1 TOPO vector in both orientations using a TOPO cloning kit (Invitrogen, P/N 46-0801). The plasmids were linearized using BamHI and 1μg of DNA used for in vitro transcription with a DIG RNA labeling mix (Roche, 11277073910) and T7 RNA polymerase (Thermo Scientific, EP0111) for 2 hours in 37°C. The reaction was stopped by addition of 2 μl of 0.2 M EDTA and the heat-denatured reaction mix used to hybridize Southern blot membranes overnight in Church's buffer (0.25 M Na-phosphate pH 7.2, 7% SDS, 1 mM EDTA). The blots were washed 3 × 30 min in wash buffer (1× SSC, 0.1% SDS) and blocked in 75 mM maleic acid, 200mM NaCl pH 7.5, 5 % skimmed milk powder for 1 hour in room temperature. The membranes were incubated with Anti-Digoxigenin-AP Fab fragment antibody (1:10 000) (Roche, 11093274910) for 2 h in room temperature, washed 2 × 15 min with blocking solution + 0.3 % Tween and further washed for 5 min with wash buffer (100 mM Tris–HCl pH 9.5, 100 mM NaCl). The signal was detected with CDP-Star chemiluminescence substrate (1:100) (Roche, 11685627001).