Quick and dirty ways to do gel extraction of DNA

[Cathal Garvey]

Hey all, just received this set of quick/dirty Gel Extraction methods from another list.

The "Poke a hole in a little eppie then spin into a big eppie" is pretty neat, because I could do that with a dremelfuge easily. Just remember to snap off the lids before spinning or they'll fly off at high speed.

As he suggests, precipitation of the DNA through some clean method is probably for the best after using one of these methods.

I can't help but think that electrophoresing DNA out of the gel fragment would be the cleanest way to get a no-agarose-residue sample for downstream applications, but I've only ever used the kits in this lab, and to save time in adapting I've ordered kits for my home lab work too for now.

---------- Forwarded message ----------
From: <novalid...@nurfuerspam.de>
Date: 19 August 2010 09:23
Subject: Re: Methods Digest, Vol 63, Issue 4
To: Adroit A <cogn...@googlemail.com>, Met...@magpie.bio.indiana.edu


Dear Adroit,

protocol would be exagerating. simpliest method is to freeze (-80 is nice) the slice thoroughly in an eppi, thaw it and spin for 5min at max speed. then use the supernatant.

cheap homemade more quantitative way is: punch a narrow hole in the bottom of a 500µl epi, insert gel slice and freeze. thaw and place small eppi in standard size eppi. spin at moderate speed for 15 min. you may add some TE, soak 10min (or do another freeze) and spin again to increase recovery.

more expensive variant: sigma sells GeneElute Agarose spin columns. place your agarose slice in one of them and spin.

For cleanup, you might do ethanol precipitation or alike

Have fun,

Wo
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[Josh Perfetto]

On Thu, Aug 19, 2010 at 6:01 AM, Cathal Garvey <cathal...@gmail.com> wrote:

I can't help but think that electrophoresing DNA out of the gel fragment would be the cleanest way to get a no-agarose-residue sample for downstream applications, but I've only ever used the kits in this lab, and to save time in adapting I've ordered kits for my home lab work too for now.

Do you or anyone know if this would work? I'm wondering if some sort of damaging reaction would occur as the DNA came in contact with the electrode, though if so perhaps an intermediate filter could prevent that from happening, with a simple wash step to remove it? Also though, I'm wondering if electrophoresis salts like borate or acetate would cause difficulties with any downstream reactions?

-Josh 

[Nima Khazaei]

Perhaps you could do it through a silica column, and then just elute with water.

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[Mackenzie Cowell]

I think this is pretty standard... isn't it called electrolution?

Mac

On Thu, Aug 19, 2010 at 4:11 PM, Josh Perfetto <jo...@snowrise.com> wrote:

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[Nathan McCorkle]

yup, borrow or find(torrent if your country doesn't enforce copyright laws) a copy of "Molecular Cloning" by J. sambrook and D. Russell:

http://books.google.com/books?id=Bosc5JVxNpkC&printsec=frontcover&dq=sambrook&source=bl&ots=edkNlDuS9Q&sig=Tc9UbwP0wBrJW0uYHaRZp3Paag4&hl=en&ei=tIBuTKuhGoP6lweEjNH3Dg&sa=X&oi=book_result&ct=result&resnum=7&ved=0CDUQ6AEwBg#v=onepage&q&f=false

Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[sgt york]

It's called the "freeze & squeeze." When I was in grad school, this
rapidly replaced the Qiagen kits in our whole department for routine
gel purification. It works pretty well, provided a somewhat reduced
yield doesn't bother you. As it's so easy to make more DNA, it's
usually more cost-effective to use more DNA and use this MUCH cheaper
protocol. IIRC, there was an article (I'm pretty sure it was an
article and not a paper) about it in Nature or Science.

There are a lot of ways to do it, and a Google of "freeze and squeeze"
will give you a lot of results. The way we used to do it by the
following protocol:

Materials:
0.5mL centrifuge tube
1.5ml centrifuge tube
Synthetic pillow stuffing (NO COTTON, that will reduce your yield
dramatically). We used the Wal-Mart store brand stuff.
Aluminum foil
Prep:
Take a 0.5mL eppendorf tube and pierce the bottom with an 18ga
needle. Obviously, you should be very, very, very careful doing this.
Slip the smaller tube inside the larger tube and put some pillow
stuffing into the smaller tube. Put in enough to loosely fill the
bottom quarter of the tube. This is your spin column.

Method:
1) Run a gel, cut the desired band out.
2) Put it on a piece of foil and set it in the freezer (-20 works just
fine, I never noticed any improvement with lN2 or -80). Freeze it
solid, takes about an hour or so, depending on your freezer..
3) Put the gel slice on top of the pillow stuffing (no need to thaw)
4) Quick spin the elute out. Should only take 30 seconds or so; don't
overspin it.
5) If desired, clean it up with a EtOH precip or dialysis disks over
buffer.

I always had great results with this technique, and it's a hell of a
lot cheaper than Qiagen kits. Less work, too.

You can experiment with sterilizing the stuffing if you want. One guy
tried sterilizing it using a UV crosslinker, but I don't recall him
getting significantly better yields than people that just precipitated
it after the spin.

[JonathanCline]

On Aug 24, 6:37 am, sgt york <jv...@yahoo.com> wrote:
>
> Materials:
>         0.5mL centrifuge tube
>         1.5ml centrifuge tube
>         Synthetic pillow stuffing (NO COTTON, that will reduce your yield
> dramatically). We used the Wal-Mart store brand stuff.

Didn't this method traditionally use a bit of glass filter instead of
pillow stuffing?


## Jonathan Cline
## jcl...@ieee.org
## Mobile: +1-805-617-0223
########################

[sgt york]

> Didn't this method traditionally use a bit of glass filter instead of
> pillow stuffing?

Yes, the original protocol used a type of fiberglass. We started off
with that, but that stuff is a pain to work with (literally,
sometimes) and we found that the pillow stuffing worked just as well.

I imagine you could also use a but of filter paper, but you'd want to
avoid anything with a charge on it or that can bind DNA (cellulose,
nylon)

[JonathanCline]

On Aug 24, 6:37 am, sgt york <jv...@yahoo.com> wrote:

> It's called the "freeze &squeeze."

Added to the FAQ! http://openwetware.org/wiki/DIYbio/FAQ/Methods

[EJ]

Yes, I did this all the time in the late 1970s and 1980s. Not sure
what the yield is compared to the new columns. Anyone done a side-by-
side? I imagine when the columns first came out, either they did one
or one was published somewhere.

[sgt york]

We did a side-by-side with freeze & squeeze and the Qiagen spin prep
years ago (2002?). It wasn't very rigorous, and it wasn't for
publication, though; just an in-house determination for cost
effectiveness after we rediscovered the technique.

The yield is somewhat down (IIRC, ~20%?), and the product is suitable
for sticky or blunt cloning (all we tried) with minimal cleanup
(precip in EtOH, wash in 70%, dry, redissolve). Some people also used
dialysis disks to clean it up. I don't think there was much
difference. We used this to make constructs for protein preps,
transgenic & knockout mice, in situ hyb, Southerns, etc.

It got to the point people only used the Qiagen kits for really small
chunks (like 50bp) or low DNA amounts (as in, you can barely see it on
the gel).

[Cathal Garvey]

I thought Qiagen kits were pretty dodgy for small fragments?

Thanks for sharing that, it's great to have a first hand account. What was your protocol? Stuffing or glass wool? :)

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