I've got two gels running while I type. Both will help me decide if the MAP7 DNA preps we have are suitable for my tweezers experiments. Both gels contain high and low concentrations of two different MAP7 DNA preps, along with a size standard consisting of intact phage lambda DNA (48.5kb) and a HindIII digest of the same DNA.
The first gel is a conventional agarose gel - the voltage is created by a pair of simple wire electrodes, one running across each end of the gel box. To increase the resolution (i.e. separation) of DNA fragments bigger than 15-20kb, the gel has a lower concentration of agarose than is usually used (0.6% rather then 0.8-1.0%). This makes it more fragile, so I'll need to handle it very carefully tomorrow when I'm photographing it. I'm also using a much lower voltage, which will also help spread out the big fragments and squeeze together the little ones I don't need to resolve.
The second gel is a "CHEF" pulsed field gel. This uses pulsing electric fields in different directions (120 degrees to each other) to jiggle the DNA fragments back and forth as they move through the gel. This forward-left then forward-right pushing has little effect on the separation of small fragments (less than about 20kb) but it greatly improves the separation of the big fragments. The new apparatus (belonging to the new lab next door) is much more sophisticated than the old one that's collecting dust on our top shelf, and I had to call technical support to learn how to dumb it down enough that I could control what it was doing.
In admiring this new apparatus we developed a new rule of thumb relating the cost of a piece of scientific equipment to the number of buttons it has. This one has more than 50 buttons, and cost about $40,000; our old one had about 7 buttons and cost $4500. This only applies to equipment that doesn't come with its own computer. The real-time PCR machine we share with other labs cost $70,000; it gets away with having only a single button because it's controlled by very complicated software.
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Whenever anyone mentions agarose gels, I point them to this just in case they haven't tried Kern and Brody's borate buffer. The easy version is this:
ReplyDelete76.34g sodium borate decahydrate
66g boric acid
qs. 2 litres in water
This is a 100mM solution, which is 20X and should have a pH of close to 7.9. Working (1X) solution, 5mM, will have a pH of around 8.5. You can run the gels at 10V/cm with little or no heat buildup; if you want to go for the 35V/cm superfast run, you may need to do a little optimizing.
Probably not much use for the expt you describe, and I don't know how it would work in pulsed field, but for routine agarose gel work I find the borate buffer much better than Tris-based ones.
Hi Bill, do you need to just mix the reagents or do you need to adjust Ph like other recipes do. Also, some recipes do not include boric acid, why do you need to include it? The other recipes where just basically mixing sodium borate tetrahydrate with water...
DeleteSo do you know anything about the chemistry of the 'breakdown of the buffer' we're often warned about for long runs? The idea seems to be that exposure to the current (voltage? heat?) changes the chemical properties of Tris. Presumably this doesn't happen with borate.
ReplyDeleteWhy is the pH of the dilute solution higher than the pH of the 20X solution? Shouldn't it be closer to the pH of water, instead of farther from it?
I was always told that long runs, by virtue of electrolytic production of H+ at one electrode and HO- at the other, altered the buffer pH and wore down its buffering capacity. Since the borate has little buffering capacity to begin with, that would seem not to be relevant. In addition, simple mixing of the two chambers should restore the initial balance, and many gel rigs actually have the two chambers connected (presumably for this purpose). I don't know anything about "buffer breakdown" or chemical changes in Tris. The big problem, of course, is heat -- we've all seen melted gels!
ReplyDeleteShouldn't it be closer to the pH of water
That sounded right at first, so -- since I'm in the lab right now -- I went and checked the pH of the 1X solution, and it's right on 8.5.
Then I thought: wait a minute, dilution reduces the concentration of all species, so the pH should move towards the pKa of the relevant acid:
pH = pKa + log [A-]/[HA]
In the case of borax/boric acid solutions, the apparent pKa is about 9.2, so the pH goes up when a solution of pH < 9.2 is diluted. I say "apparent pKa" because borate is a complicated little beastie.