Mitochondrial DNA isolation

Jim Cummins cummins at POSSUM.MURDOCH.EDU.AU
Sat Jun 19 22:52:53 EST 1993

The following should help.  We isolate mtDNA from sperm and testis biopsy
material by homogenising in SDS lysis buffer (10 mM Tris pH 8.0, 10mM
EDTA, 50 mM NaCl, 2%SDS, 10ulproteinase K (10mg/ml) for 1.5 hours at
56C and purify using Prep A Gene (Bio Rad).  Remove tissue by
centrifuging and incubate for 1 hour further at 56C

Check out the following refs. Zhang et al (1992) FEBS 297: 34-38 Torii et
al (1992) Am J Respir Cell Mol Biol 6:543-549 Cortopassi $ Arnheim (1990)
NAR 18(23): 6927-6933 Miquel (1991) Arch Gerontol Geriatr 12: 99-117
Linnane et al (1992) Mutation Res 275: 195-208 Linnane et al (1992) In
Adenine Nucleotides in Cellular Energy Transfer and Signal Transduction, S
Papa, A Azzi and JM Tager (eds) Birkhauser Verlag, Basel/Switzerland, pp
137-149.  This last also contains information on mtDNA deletions in rats
with aging and uses an interesting cell model lacking mtDNA. Leprat et al
(1990) in Mechanisms of Ageing and Development 52: 149-167 have described
a technique for monitoring cell ageing and mitochondrial function using
fluorescent probes. 
Let me know how you go. What aspect of oxidative damage are you interested in?
Jim Cummins

Dr Jim Cummins                            +61-9-360 2668
School of Veterinary Studies          FAX =61-9-310 4144     
Murdoch University               cummins at
Murdoch, Western Australia 6015

On 18 Jun 1993, Christoph Schller wrote:

> Dear Netters
> Does anyone have a protocol to isolate mt-DNA from tissue (rat
> brain)? How to estimate nuclear DNA contamination? We want to analyse
> oxidative damage.
> Christoph Schueller.
> In der Kuerze liegt die Wuerze.

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