When isolating mitochondria for mtDNA, the protocol we are using says that after obtaining mitochondrial pellet (after pooling in a cytosol buffer), to rehydrate the mitochondria in PBS. Is this PBS (rehydration) step necessary (or beneficial) if all one wants to do is isolate the mtDNA using SDS/ProtK?
Why are you using rehydration methods instead of proceeding straight from fresh mitochondria by ancient methods.
The protocol we are using implies that the "cytosol buffer" used for isolation (from Active Motif kit) dehydrates the mitochondria. I obtained some mito DNA directly from the fresh (initial) pellet, but there was a PBS rehydration step (I assumed for other uses such as protein isolation, mito activity assays, etc.), so I was just wondering if perhaps there would be a better yield with the rehydration step. The protocol uses the buffer from the kit, dounce homog., and a series of centrifugation steps (2 initial, 1 quick spin at full speed, then 20 min at 3000 rpm, then four spins of the remaining sup) to obtain 4 successive mito pellets (i.e., four 11,000 rpm, 20 min spins in microfuge, respinning sup) that are then combined . This is an isolation from fresh mouse liver. Thanks!
Often for functional assays mitochondrial preparations are very concentrated with paste like consistency. One need much diluted sample for Prot-K/SDS preparation. You may even use Tris-NaCl for dilution if you only intend to prepare DNA or one of many DNA isolation kit.
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