DNA from muscle (1993 version...)

Paulo Magalhaes pamaga at biobase.aau.dk
Tue Jan 5 05:25:21 EST 1993


Last year (!) I asked for some help on DNA extraction from muscle
biopsies. I got some answers, but I'm afraid no real help. So, for
the time being anyway, I'll stick to my present method.

I did, however, get a number of requests for my method. To save me
the trouble of replying individually, I thought it would be a good idea
to post it here. I have to apologise, but my knowledge of
mainframes and the likes is not what I would like it to be... Perhaps
what follows is readable (modems, tab spaces and various other
gremlins always play tricks on me...); any questions, doubts, etc,
just drop me a line.

Again I would like to reiterate my request. If there's anyone out there
who knows of a faster/simpler/more efficient method, I'd very much
like to hear about it. My interest is only in mtDNA, by the way.

Regards from Denmark.

Rigshospitalet 4062
Copenhagen - Denmark



Day 1

	1)	Rinse homogenisers in H2O and soak in 0.1 M HCl for 30'.
	2)	Rinse in H2O, ethanol and let dry.

	3)	Thaw biopsy and transfer to homogeniser
	4)	Add 1 ml of DB and homogenise well.

	5)	Transfer homogenate to an epp.
	6)	Add 25 ul of 20% SDS
	7)	Add 20 ul of 20 mg/ml ProK
	8)	Mix well and incubate O/N at 370C.

	9)	Rinse homogenisers in H2O and soak O/N in 20% Extran.

Day 2

	0)	This is just a reminder to wash the homogenisers and store them away...

	1)	Divide the contents of the O/N digestion into 2 equal portions.

	2)	Add 500 ul of PhOH and vortex briefly.
	3)	Add 500 ul of Chlorof. and vortex briefly.

	4)	Spin for 3'.

	5)	Remove 530 ul of the aqueous phase and transfer it to a new eppendorf.
	6)	Add 530 ul of Chlorof. and vortex briefly.

	7)	Spin for 3'.

	8)	Transfer 430 ul of the aqueous phase to a new eppendorf tube.
	9)	Add 215 ul of 7.5 M NH4Ac.
	10)	Add 860 ul of 100% EtOH.
	11)	Mix gently but thoroughly (keep at 40C if necessary)

	12)	Spin for 15'.
	13)	Remove supernatant carefully.

	14)	Add 1 ml of 70% EtOH.

	15)	Spin for 15'.
	16)	Remove supernatant carefully and dry under a lamp (30 - 40')

	17)	Dissolve the DNA in 50 5l of T-4E.


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