Ooh, controversy.
I got my Honours student ('W') to perform the experiment yesterday, and this is just in:
The above is a photograph of an agarose gel, which is used to separate different fragments of DNA. It has been stained with ethidium bromide, which glows under ultraviolet light when bound to DNA.
Legend:
M: Markers
1: Uncut DNA
2: Squeezed DNA (not frozen)
3: Freeze-squeezed DNA
I did not directly supervise W while he did this: I gave him the method exactly as written and told him to get on with it. He took a gel slice, cut it in two, froze one bit and then spun both pieces through the Spin-X.
Conclusion: If you cut your plasmid DNA with a restriction enzyme or two, and gel-purify it, it helps to freeze the agarose slice before trying to extract it (note that lane 2 is empty, and there's a lovely band in lane 3).
Alethea, I'd be interested to read your take on this.
Comments
My take is that you definitely don't have a band in 2 and do in 3. (As an aside, why does your uncut ride *lower* than your cut plasmid? I know you're going to say "supercoiled" but we usually get the opposite... now you can say "concatamers"! That's how we explain everything - one or the other)
Hey, you know what? I was thinking of our purifying out bands from polyacrylamide gels (we needed to separate 26bp from 40bp). The rare times I've had to purify from agarose gel I've also been a "column whore" - or at least a silica-based one (that implies I'm stacked, which is not the case) - having employed either Qiaquick columns or Glassmilk for these purposes. Sorry for the mix-up.
But I'm still curious what the freezing does. Breaks the sugars better?
Posted by: Alethea | March 9, 2007 08:16 PM
supercoiled plasmid runs faster than linearized; although nicked, circular plasmid will sometimes run slower than linearized.
the freezing probably works by the formation of water ice crystals in the gel. this disrupts the organization of the agarose matrix - effectively making the pores larger, letting the DNA elute out easier.
Posted by: mw | March 12, 2007 08:25 AM
Well, I'm convinced (n=1, good enough for me!). Doesn't really tell us how efficient the freeze-squeeze method is though.
Not that it matters, as long as you get "enough".
Alethea - that "stacked" pun were absolutely horrible, it were. Well done.
Posted by: Whiffling while frozen | March 12, 2007 09:49 AM
mw — Yes; if you follow the instructions in the plasmid prep *kits* (bleh) or the manual instructions in Maniatis etc. you get nicked DNA. There was a paper about this in, oh, '97?
Essentially, all the methods say use fresh NaOH/SDS and incubate for 5 - 10 minutes, but this is complete poppycock. It nicks your DNA. Old NaOH/SDS and no incubation (but keeping it cold) is much better.
Posted by: BK | March 12, 2007 10:23 AM
I like using either Qiaex II beads or glassmilk to extract DNA but I modify the Qiaex protocol thus:
Instead of adding the beads and buffer to the gel slice and mixing for however long they say, I first melt the agarose at 55-60 C for ~30 minutes, then add the beads and incubate on the rocker/rotator at RT for at least one hour. You get excellent recovery this way, much better than with the Qiaex II protocol. Also, because I am lazy, I tend to leave the cut gel slice at 4 C overnight before extraction, which might help!
Posted by: Joolya | March 20, 2007 06:23 AM