Extracting DNA as a fiber/strand - Dumb question! Need practical help. (Oct/30/2006 )

I'm a crystallographer, and I've worked with proteins for two years now, but an old problem that I am yet to solve is extracting DNA as a fibre. My background isn't biochem, it's more x-ray, so I am pretty confused when it comes to reading biochem/mol bio protocols.

All the protocols mention DNA precipitation by ethanol, but understandably, none of the authors cares about getting it out as a strand. Supposedly it's an easy elementary school experiment to put DNA into a solution, then add ethanol/isopropanol and viola, strands come to the water/ROH layer, and you can pull them out with a glass rod?

I have DNA I prepared with the Qiagen kit, and I have a lot more of this fish sperm(gross) DNA from Spectrum:

http://www.spectrumchemical.com/retail/pro...ct%5Fid=3994248

Any suggestions on what I can do to pull a strand from that and some combination of salt, ethanol, isopropanol and solution would be greatly appreciated. Some of the older papers I am reading mention some complicated gel stuff, as if I'm not already confused! Argh!

Any help would be greatly appreciated, thank you!

Clarification: I am trying to get a thick strand, i.e a physical strand, like physical string, for physical purposes, not a single molecule as for microbio/bio purposes.

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I've used Qiagen kits to prepare genomic DNA before, and I found that the DNA is sometimes degraded. This could be why you can't get good long strands from your DNA when you precipitate it. If you can, prepare your DNA by phenol/chl extraction. Be sure you never vortex the DNA to, prevent shearing, just shake by hand to mix.

I actually don't spool my DNA but my protocol tells how to:

My DNA is from mouse tails and I extract once with phenol and once with phenol/chl/isoamyl alcohol.
Then add 1 ml of 100% EtOH (-20 degrees) and 50 ul of 3M sodium acetate (pH 6). My protocol says the sodium acetate helps the DNA adhere to the glass when spooling.

Now what I do is just invert the tube several times and I can start to see the stringy DNA precipitate as I invert it more it forms a tighter clump. At this point the protocol says to spool the DNA around a glass micropipet (from VWR) whose ends have been sealed with a flame. You will have to play around with the spooling process, but it seems like it would work after inverting once and then spooling.

Then you wash the DNA once or twice with 70% EtOH.

Then it says to break the end of the capillary tube into a microfuge tube and add TE and put at 65 degrees for 5 min to dissolve.

My final amount of DNA is ~20 ug dissolved in 200 ul of TE, so I would try to have this amount so you know you can see the precipitate.

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for the spooling...I think you get strands that are easier to see if you use isopropanol; then later the washes happen with ethanol

as for the spooling tool...I find that if you hold the pasteur pipet tip in a bunsen flame - it only takes a couple of seconds- at about 45 degree angle, the tip will melt and fall down a bit...it's hard to describe, but you'll see what I mean if you try it the end result is shaped like a shepherd's crook and the end is sealed. the curve/hook shape at the end makes it easier for the spooled strands to stay on the tool when you remove it from the solution (DNA is very slippery)

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Thanks for the tips. My problem is getting the strand DNA in the first place I'm sure it's just a stupid error that the biochem whizzes would know better than I.

One time, I squirted DNA in TE buffer out of a pipette into a 50mL tube circling around, and whatever I squirted seemed to make a very very narrow fiber, thinner than spiderwebs.

When I just leave the DNA in water solution and pour isopropanol over it, nothing happens.

Once I dissolved it in water, added NaCl to saturation, and then poured a layer of isopropanol, and these weird granules began to rise up to the water/R-OH boundary.

So I know I'm close, but I have no idea on how to make the DNA precipitate as a strand.

Right now I have 5 grams of "Deoxyribonucleic Acid Not highly polymerized. Source: Fish sperm.", not in salt, and it comes as a powder. Do I just suspend this in TE buffer, then cool, add some salt, and then pour a layer of isopropanol on top, and wait for the strands to form?

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You'll need to go buy an onion, but check this out:
http://www.carolina.com/biotech/onion.asp

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I am sure with Qiagen kits or any kits that use columns, DNA is sheared to about 20 kb so its unlikely you will pull it out as a strand. In my experience, you need to ensure minimal shearing of the DNA, so not too much pipetting or vortexing etc.

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Ahhhhhh ok. So if I got DNA as a powder, I'm guessing that won't get pulled as a strand either?

Thanks phage, I was trying to avoid doing the dirty work of extraction too though

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Does it matter where the DNA comes from? I have a pretty easy method for extraction of nucleic acids from bacteria. When you precipitate with isopropanol, I always get the stringy precipitation of nucleic acids which can then be spooled out. Let me know so I can post the reference for you.

M.

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Nope, it doesn't matter to me, I'm planning on shooting x-ray beams through it and I've only been trying different things out of desparation! Thanks!

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OK..here's a quick recipe. Make XS buffer:

0.5 g Potassium ethyl Xanthogenate (Fluka 140-89-6)
10 mL 4M Ammonium acetate
5 mL 1M Tris-HCl pH 7.4
2 mL 0.45M EDTA
2.5 mL 20 % SDS
Water to 50 mL

Pellet 1-2 ml of bacterial culture in a 2 mL eppendorf
Remove supernatant and resuspend pellet in 1 ml XS buffer
Incubate @ 70 degrees for 1 hour
Vortex tube for 10 sec and incubate on ice for 30 min
Centrifuge at 14, 000 rpm for 10 minutes
Carefully remove supernatant to a new tube and add 1 volume of isopropanol
Invert tube a few times, DNA will precipitate as a thread
spool out with a tip or tooth pick
Wash spooled DNA in a tube containing 70 % ethanol
Resuspend DNA in 200 ul of dH20

RNA will be present so you may want to Rnase treat your sample.

This has been taken from:

XANTHOGENATE NUCLEIC ACID ISOLATION FROM CULTURED AND ENVIRONMENTAL CYANOBACTERIA

Tillett D. & Neilan B.A.
Journal of Phycology, Volume 36, Number 1, February 2000, pp. 251-258(8)

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