1. Commonly used buffers for molecular cloning
TE(pH7.4)
10mmol/L Tris.Cl(pH7.4) 1mmol/L EDTA(pH8.0)
TE(pH7.6)
10mmol/L Tris.Cl(pH7.6) 1mmol/L EDTA(pH8.0)
TE(pH8.0)
10mmol/L Tris.Cl(pH8.0) 1mmol/L EDTA(pH8.0)
STE (or TEN)
10mmol/L Tris. Cl (pH8.0) 1mmol/L EDTA (pH8.0) 0.1mol/L NaCl
STET
10mmol/L Tris.Cl(pH8.0) 1mmol/L EDTA(pH8.0) 0.1mol/L NaCl 5%TritonX-100
TNT
10mmol/L Tris.Cl(pH8.0) 150mmol/L NaCl 0.05%Tween20
2. Phosphate buffer
(1) Preparation of 0.1 mol/L potassium phosphate buffer at 25°C※
pH | 1mol/LK 2 HPO 4 (ml) | 1mol/L KH 2 PO 4 (ml) |
5.8 | 8.5 | 91.5 |
6.0 | 13.2 | 86.8 |
6.2 | 19.2 | 80.8 |
6.4 | 27.8 | 72.2 |
6.6 | 38.1 | 61.9 |
6.8 | 49.7 | 50.3 |
7.0 | 61.5 | 38.5 |
7.2 | 71.7 | 28.3 |
7.4 | 80.2 | 19.8 |
7.6 | 86.6 | 13.4 |
7.8 | 90.8 | 9.2 |
8.0 | 94.0 | 6.2 |
(2) Preparation of 0.1 mol/L sodium phosphate buffer at 25°C※
pH | 1mol/L Na 2 HPO 4 (ml) | 1mol/L NaH 2 PO 4 (ml) |
5.8 | 7.9 | 92.1 |
6.0 | 12.0 | 88.0 |
6.2 | 17.8 | 82.2 |
6.4 | 25.5 | 74.5 |
6.6 | 35.2 | 64.8 |
6.8 | 46.3 | 53.7 |
7.0 | 57.7 | 42.3 |
7.2 | 68.4 | 31.6 |
7.4 | 77.4 | 22.6 |
7.6 | 84.5 | 15.5 |
7.8 | 89.6 | 10.4 |
8.0 | 93.2 | 6.8 |
※: Dilute the two mixed 1 mol/L stock solutions to 1000 ml with distilled water and calculate the pH value according to the Henderson-Hasselbalch equation:
pH = pK' + 1g ([proton acceptor] / [proton donor])
Here, pK'=6.86 (25°C).
3. Electrophoresis buffer
Sequencing gel loading buffer
98% deionized formamide
10mol/L EDTA (pH 8.0)
0.025% Xylene Cyanol FF
0.025% bromophenol blue
Formamide: Many batches of reagent grade formamide have a purity that meets the requirements for use and do not require further treatment. However, once it is slightly yellow, it should be deionized by stirring the formamide with Dowex XG 8 mixed bed resin on a magnetic stirrer for 1 hour, and filtered twice with Whatman No. 1 filter paper. The deionized formamide is divided into small portions and stored at -70°C filled with nitrogen.
Commonly used electrophoresis buffers
Buffer | Use liquid | Concentrated stock solution (per liter) |
| Tris-acetic acid (TAE) | 1×: 0.04 mol/L Tris-acetic acid | 50×: 242g Tris base |
| 0.001mol/L EDTA | 57.1 ml glacial acetic acid | |
| 100ml 0.5mol/L EDTA (pH8.0) | ||
| Tris-phosphoric acid (TPE) | 1×: 0.09 mol/L Tris-phosphate | 10×: 10g Tris base |
| 0.002mol/L EDTA | 15.5ml 85% phosphoric acid (1.679g/ml) | |
| 40ml 0.5mol/L EDTA (pH8.0) | ||
| Tris-boric acid (TBE) a | 0.5×0.045mol/L Tris-boric acid | 5×: 54g Tris base |
| 0.001mol/L EDTA | 27.5 Boric acid | |
| 20ml 0.5mol/L EDTA (pH8.0) | ||
| Alkaline buffer b | 1×: 50mmol/L NaOH | 1×: 5ml 10mol/L NaOH |
| 1mmol/L EDTA | 2ml 0.5mmol/L EDTA (pH8.0) | |
| Tris-Glycine c | 1×: 25mmol/L Tris | 5×: 15.1 g Tris |
| 250mmol/L Glycine | 94g Glycine (electrophoresis grade) (pH 8.3) | |
| 0.1% SDS | 50ml 10% SDS (electrophoresis grade) |
illustrate:
①TBE solution will form precipitate after long-term storage. To avoid this problem, 5× solution can be stored in a glass bottle at room temperature and discarded after precipitation occurs.
In most cases, 1×TBE is used as the working solution (i.e., a 1:5 diluted concentrated stock solution) for agarose gel electrophoresis. However, 0.5× working solution has sufficient buffer capacity. Currently, almost all agarose gel electrophoresis uses a 1:10 diluted stock solution as the working solution.
The buffer tank for polyacrylamide gel vertical trough is smaller, so the current passing through the buffer is usually larger, and 1×TBE is required to provide sufficient buffer capacity.
②Alkaline electrophoresis buffer should be prepared before use.
③SDS polyacrylamide gel electrophoresis using Tris-glycine buffer.
2×SDS gel loading buffer:
100mmol/L Tris·HCl(6.8)
200mmol/L dithiothreitol (DTT)
4% SDS (electrophoresis grade)
0.2% bromophenol blue
20% glycerol
2×SDS gel loading buffer without DTT can be stored at room temperature. 1 mol/L storage solution should be added to the above buffer before use.
4. Gel loading buffer
Buffer type | 6× Buffer | Storage temperature |
| 0.25% bromophenol blue |
|
| 0.25% Xylene Cyanol FF | ||
| 40% (W/V) sucrose aqueous solution | ||
| 0.25 bromophenol blue |
|
| 0.25% Xylene Cyanol FF | ||
| 15% Polysucrose (Ficoll400) | ||
| 0.25% bromophenol blue |
|
| 0.25% Xylene Cyanol FF | ||
| 30% glycerol aqueous solution | ||
| 0.25% bromophenol blue |
|
| 40% (W/V) sucrose aqueous solution | ||
| Alkaline loading buffer: | ||
| 300mmol/L NaOH | ||
| 6mmol/L EDTA | ||
| 18% Polysucrose (Ficoll400) |
|
| 0.15% bromocresol green | ||
| 0.25% Xylene Cyanol FF |
The above gel loading buffer has three purposes: to increase sample density; to ensure that DNA enters the sample well evenly; to make the sample appear colored, so that the loading operation is more convenient, and to contain dyes that can migrate toward the anode at a predictable rate in the electrophoresis block. The rate at which bromophenol blue migrates in agarose is about 2.2 times that of xylene cyanol FF, regardless of agarose concentration. When 0.5×TBF is used as the electrophoresis buffer, the migration rate of bromophenol blue in agarose is about the same as that of 300bp long double-stranded linear DNA, while the migration rate of xylene cyanol FF is the same as that of 4kb long double-stranded linear DNA. In the range of agarose concentration of 0.5% to 1.4%, these corresponding relationships are not significantly affected by changes in gel concentration.
The choice of dye to use is a matter of personal preference, but for alkaline gels, bromocresol green should be used as the tracking dye because it is a more blue color than bromophenol at alkaline pH.
5. Preparation of Tris buffer with various pH values
Preparation of Tris buffer with various pH values | |
Required pH value (25℃) | Volume of 0.1 mol/L HCl |
7.1 | 45.7 |
7.2 | 44.7 |
7.3 | 43.4 |
7.4 | 42.0 |
7.5 | 40.3 |
7.6 | 38.5 |
7.7 | 36.6 |
7.8 | 34.5 |
7.9 | 32.0 |
8.0 | 29.2 |
8.1 | 26.2 |
8.2 | 22.9 |
8.3 | 19.9 |
8.4 | 17.2 |
8.5 | 14.7 |
8.6 | 12.4 |
8.7 | 10.3 |
8.8 | 8.5 |
8.9 | 7.0 |
To prepare a 0.05 mol/L Tris buffer at a specific pH value: Mix 50 ml of 0.1 mol/L Tris base solution with the corresponding volume (in ml) of 0.1 ml/L HCl shown in the table above, and add water to adjust the volume to 100 ml
(2) Effect of temperature on pH value of 50 mmol/L Tris·HCl solution
4℃ | 25℃ | 37℃ |
8.1 | 7.5 | 7.2 |
8.2 | 7.6 | 7.3 |
8.3 | 7.7 | 7.4 |
8.4 | 7.8 | 7.5 |
8.5 | 7.9 | 7.6 |
8.6 | 8.0 | 7.7 |
8.7 | 8.1 | 7.8 |
8.8 | 8.2 | 7.9 |
8.9 | 8.3 | 8.0 |
9.0 | 8.4 | 8.1 |
9.1 | 8.5 | 8.2 |
9.2 | 8.6 | 8.3 |
9.3 | 8.7 | 8.4 |
9.4 | 8.8 | 8.5 |
(3) pKa values of commonly used buffers
Buffer | Molecular weight | pKa value | Buffer range |
Tris a | 12.1 | 8.08 | 7.1～7.9 |
HEPES b | 283.3 | 7.47 | 7.2～8.2 |
MPOS c | 209.3 | 7.15 | 6.6～7.8 |
PIPESd | 304.3 | 6.76 | 6.2～7.3 |
MES | 195.2 | 6.09 | 5.4～6.8 |
a: tris(hydroxymethyl)aminomethane; b: N-2-hydroxyethylpiperazine-N'-2-ethylphosphonic acid; c: 3-(N-morpholino)propanesulfonic acid; d: N,N'-bis(2-ethanesulfonic acid)piperazine; e: 2-(N-morpholino)ethanesulfonic acid.
7. Effect of temperature on pH of commonly used buffer solutions
Buffer system | pKa(20℃) | △pKa/10℃ |
Mes | 6.15 | -0.110 |
Ada | 6.60 |
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