Making a working buffer from a concentrate
Most buffers are stored as a concentrate and diluted for use. The volume of concentrate you need is the final volume divided by the fold:
Vstock = Vfinal ÷ fold so 1 L of 1× from 50× = 1000 ÷ 50 = 20 mL of stock + 980 mL water
Choose the working strength above and the table gives the amounts from powder; the note under it gives the equivalent dilution of the stock. For any other dilution use thedilution calculator; to design a buffer from a pKa rather than a recipe, the Henderson–Hasselbalch andphosphate buffer tools do that, and themolarity calculator converts any molar target to grams.
TAE or TBE for agarose gels?
Both are Tris–EDTA buffers that differ in the acid. TAE (acetate) has lower buffering capacity and runs out during long electrophoresis, but it resolves large fragments better and does not inhibit ligases and polymerases, so it is the choice when the DNA will be cut out of the gel. TBE (borate) buffers more strongly, gives sharper bands for fragments under about 2 kb and heats less, but borate binds to agarose and to DNA, and carries over into enzymatic reactions. Use the same buffer in the gel and in the tank. Theagarose gel calculator gives the agarose for a given percentage and volume.
Why PBS is pH 7.4
PBS mimics the ionic strength and pH of extracellular fluid, about 300 mOsm and pH 7.4, so cells neither swell nor shrink in it. The phosphate pair, monobasic and dibasic, buffers around its pKa of 7.2. The salt keeps the solution isotonic; without it, plain phosphate buffer would lyse cells. PBS contains no Mg²⁺ or Ca²⁺ in the standard recipe, which is why it is used to wash and detach adherent cells; buffers for cell culture that must keep cells attached use DPBS with calcium and magnesium.
Adjusting pH, and the Tris temperature effect
Adjust pH after the components are dissolved but before the final volume is reached, then top up with water. Use concentrated acid or base so the volume added is small. The Tris recipes above list approximate acid volumes; the exact amount depends on the batch and the water, so always finish with a meter. Tris is unusual in that its pKa drops by about 0.03 units per degree, so a Tris buffer set to pH 8.0 on the bench reads about 8.6 in the cold room and 7.7 in a 37 °C incubator. Set the pH at the working temperature, or at least let the solution cool to room temperature before adjusting, since dissolving Tris warms it.
Storage and sterilisation
Salt and buffer stocks can be autoclaved at 121 °C for 20 minutes and then keep for months at room temperature. Do not autoclave solutions containing detergents, glycerol-rich sample buffers, reducing agents or antibiotics; filter-sterilise those where sterility matters. Concentrates of TBE and PBS precipitate in the cold and are best stored at room temperature. Media should be autoclaved as soon as they are made and any additives, such as antibiotics, added only after cooling below 50 °C. Label every bottle with the buffer, strength, pH and date.
Concentrates and their working dilutions
| Stock | Working | Dilution |
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| 10× PBS | 1× PBS | 100 mL per litre |
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| 50× TAE | 1× TAE | 20 mL per litre |
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| 5× TBE | 1× or 0.5× TBE | 200 or 100 mL per litre |
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| 10× TBS | 1× TBS or TBS-T | 100 mL per litre, plus 1 mL Tween 20 for TBS-T |
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| 10× Tris-glycine-SDS | 1× running buffer | 100 mL per litre |
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| 10× Tris-glycine | 1× transfer buffer | 100 mL plus 200 mL methanol per litre |
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| 20× SSC | 2× or 1× SSC | 100 or 50 mL per litre |
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| 4× Laemmli | 1× in the sample | 1 part buffer to 3 parts sample |
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Frequently asked questions
How do I make 1× TAE from 50× stock?
Dilute it 1 in 50: 20 mL of 50× TAE plus water to 1 L. In general, the volume of concentrate is the final volume divided by the fold, so 1 L of 1× from a 10× stock needs 100 mL of stock and 900 mL of water.
What is the difference between TAE and TBE?
TAE uses acetate, TBE uses borate as the buffering ion. TBE has higher buffering capacity and gives sharper bands for small fragments and long runs, but borate inhibits some enzymes, so DNA to be purified from a gel is better run in TAE. TAE is also the buffer for large fragments and for lower resistance during long runs.
Why does my 10× PBS have a precipitate?
The phosphates are close to their solubility limit in the concentrate, especially in the cold. Warm the bottle and stir until it clears before diluting. If it will not clear, make it fresh. Storing 10× PBS at room temperature avoids the problem.
Do I need to adjust the pH of Tris-glycine running buffer?
No. The ratio of Tris to glycine sets the pH at about 8.3 by itself, and adding acid or base introduces extra ions that distort the gel. The same is true of TAE and TBE: mix the components and check the pH, but do not adjust it.
Why does the pH of Tris buffer change with temperature?
The pKa of Tris falls by about 0.03 units for every degree the temperature rises. A buffer set to pH 8.0 at 25 °C is about pH 8.6 at 4 °C and about 7.7 at 37 °C. Set the pH at the temperature the buffer will be used, and let a warm solution cool before the final adjustment.