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I would like to run the following protocol to detect plasmid presence in bacteria. Would be possible to replace 'Tris- acetate' with just Tris?

We don't have 'Tris- acetate' in the lab. What is the role of acetate here?


Hot alkaline method for all plasmid sizes and bacteria (Kado & Liu,1981), modified

  • Centrifuge 2-3 ml of culture, resuspend pellet in 1 ml of solution containing 0.04 M Tris- acetate, pH 8.0 (adjust pH with glacial acetic acid) and 2 mM EDTA

  • Add 2 ml of lysis buffer (0.05 M Tris, 3% SDS, pH 12.50, adjusted with 2 N NaOH) and mix

  • Incubate at 60-68°C for 30-45 min (strain dependent)

  • Add to hot samples 6 ml of phenol/chloroform (1:1) and mix gently to complete emulsification

  • Separate phases by centrifugation at 10.000 x g for 15-20 min at RT and transfer the upper aqueous phase carefully (avoid interphase which contains debris) to new tube containing 1 volume of chloroform. Mix and centrifuge again for separation of phases

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  • $\begingroup$ You should be able to add glacial acetic acid to Tris to produce Tris-acetate. $\endgroup$ – f'' Jul 11 '16 at 12:54
  • $\begingroup$ How much glacial acetic acid @f'' $\endgroup$ – biotech Jul 12 '16 at 6:48
  • $\begingroup$ @biotech Enough to make the pH 8.0, as your procedure says. $\endgroup$ – f'' Jul 12 '16 at 14:32
  • $\begingroup$ Ok @ f'', I had a misunderstanding with this step. Seems is not a mixture of 'Tris- acetate' salt what I have to add, but Tris and then enough acetate (glacial acetic acid) to reach pH 8.0, rigth? $\endgroup$ – biotech Jul 12 '16 at 15:49
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"Tris" simply means "three" and is used in a number of different contexts. However, in biochemistry it's normally used to refer to the common buffering agent tris(hydroxymethyl)aminomethane. You have to be a little careful, though, as there are other uses of "Tris" in biology, which may not refer to tris(hydroxymethyl)aminomethane. (For example, there's two other buffers called "bis-tris methane" and "bis-tris propane", each of which have very different properties from tris(hydroxymethyl)aminomethane.)

Your situation is referring to the standard tris(hydroxymethyl)aminomethane, though. However, when specifying a buffering system, it's often insufficient to specify just the buffering agent used. A buffer consists of a weak acid or weak base in near-equal concentration to its conjugate base or acid. Tris is the weak base being used, but to have a decent buffer you need to also have the conjugate acid form.

As this conjugate acid is charged, there's an obligate counter ion. Different buffering systems may use different counter ions. That's what the acetate is doing here - acting as a counter ion for the conjugate acid of tris(hydroxymethyl)aminomethane.

That's what "Tris-acetate" is specifying. It's using a Tris buffering system with an acetate counter ion. This buffering system is normally obtained by using enough Tris base to get the desired concentration, and then slowly adding enough acetic acid to obtain the desired pH.

Alternatively, you could use something like the Henderson–Hasselbalch equation to calculate the appropriate proportion of base and conjugate acid for your desired pH, and then specifically mix those two forms together. This isn't typically done with Tris-acetate, as the acetate counter ion form of Tris isn't commonly sold. However, Tris hydochloride is, so if you had a system which used a chloride counter ion, you could use this approach. (Though in practice you'll want to aim a little on the basic side, and adjust to the desired pH by titration - the numbers which come out of the Henderson–Hasselbalch equation are theoretically pure, and don't account for non-ideal deviations in behavior and things like weighing errors.)

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By Tris you most likely mean Tris base. Make sure that the formula is C4H11NO3 and the cas number is 77-86-1. Both should be on a label. If this is the case - you can use glical acetic acid. Slowly mix 121.14 g of Tris in 350 mL of water with 60 g of glical acetic acid. Bring volume to 500 mL. This will give you 2M solution of Tris-Acetate.

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