I wish to isolate mtDNA from cultured human cells. I am using Dounce homogenisation (glass teflon potter) in a sucrose buffer to break open the cells and further centrifuge to obtain mitochondria. I wish to isolate mitochondrial DNA from this pure mitochondrial suspension. What kind of lysis buffer or detergents should I use?
Hello Vikas,
you could use this buffer (according to Bender et al., Mol. Biol. Cell 2011):
0.5% Triton X-100; 30 mM Tris, pH 7.4; 200 mM KCl; 5 mM EDTA; 0.5 mM phenylmethylsulfonyl fluoride [PMSF]; protease inhibitors
Regards, Christian
Avoid protease inhibitor and PMSF if you schedule to do a protein digestion to remove protein and keep only nucleic acid. PMSF is a protease inhibitor too.
We use a standard lysis buffer suitable also for total cellular DNA extraction: 20 mM Tris pH 7,4, 75 mM NaCl, 10 mM EDTA and either 0,4% SDS or 1% Triton. Proteinase K digest at max 37°C and afterwards phenol/chlrooform extraction and ethanol precipitation.
Thank you very much to all of you for the inputs.
Just a clarification Christian : Why do we need PMSF or protease inhibitors if we wish to isolate nucleic acids?
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