organism: Badhamia polycephala
description: Plasmodial strain M3CVIII (kindly provided by Dr. Mark Adelman) was grown as macroplasmodia at 26*C in semi-defined medium.  Macroplasmodia were harvested directly into ice cold BSS [10 mM Tris-HCl (pH 7.5)/ 0.25 M sucrose], with all subsequent steps carried out at 4*C.  Cells were lysed in a Waring blender using two 15 second bursts at half maximum speed.  The homogenate was filtered prior to pelleting the nuclei and remaining cell debris by centrifugation for 5 min at 700xg.  Mitochondria were pelleted by centrifugation for 5 min at 5800xg, resuspended in BSS, layered over Percoll step gradients (densities 1.044-1.095 g/ml in 1 mM Tris-HCl (pH 7.5)/ 0.25 M sucrose), and centrifuged at 47,800xg for 30 seconds.  Mitochondrial fractions were collected from each gradient, diluted slowly with 2.5 volumes of 1 mM Tris-HCl (pH 7.5)/ 0.25 M sucrose, and pelleted by centrifugation at 7,600g for 5 min.  Total mitochondrial RNA was isolated using TRIzol reagent (Invitrogen) as specified by the manufacturer.  Residual DNA was removed by digestion with DNaseI (Roche) in the supplied buffer.