Preparation of Tris (hydroxymethyl) aminomethane Tris saturated phenol
Tris (tris(hydroxymethyl)aminomethane) as a commonly used biological buffer has a very wide range of applications in the field of biochemical engineering, such as nucleic acid purification, and saturated phenols have two kinds in nucleic acid purification, one is Tris saturated phenol , Also known as basic phenol, Tris balanced phenol (Tris saturated phenol or Tris balanced phenol), is a redistilled phenol saturated with Tris-Cl pH8.0, which is a light yellow transparent liquid with an irritating smell, and the upper layer is Tris Hydrochloric acid buffer solution, antioxidant 8-hydroxyquinoline is added to the solution. It is suitable for removing proteins from nucleic acid samples and extracting DNA. The other is acidic phenol, which is water-saturated phenol, used to extract RNA.
Desheng location and biological buffer products
Tris saturated phenol generally has a pH greater than 7.8. Among them, phenol is a strong protein denaturant and can denature and precipitate proteins in cells or tissues. The main role of Tris is to prevent phenol oxidation. If the phenols are oxidized, quinones (two benzene rings) will be formed. The quinones contain strong free radicals and will destroy the nucleic acid structure. At the same time, because the PH value is greater than 7, DNA is in the aqueous phase and RNA is in the organic phase in an alkaline environment. Thus, the supernatant is separated and DNA can be obtained.
Phenols on the market contain oxides such as quinones. These products can cause the cleavage of phosphodiester bonds and lead to cross-linking of RNA and DNA. They should be re-evaporated at 160°C with a condenser. Re-distilled phenol was added with 0.1% 8-hydroxyquinoline (as an antioxidant), and repeated with equal volume of 0.5mol/L Tris·Cl (pH8.0) and 0.1mol/L Tris·Cl (pH8.0) buffer Extraction makes it saturated and makes its pH value reach above 7.6, which is Tris saturated phenol (about pH7.8).
Tris saturated phenol configuration:
1. Take out the redistilled phenol in the refrigerator, put it at room temperature to a 68-degree water bath to dissolve, do not immediately put it in a 68-degree water bath to prevent the glass from bursting
2. Add 8-hydroxyquinoline to 0.1% and β-mercaptoethanol to 0.2% (original solution 14. 4MOL/ML), mix well, the solution appears yellow, and pour into a separating funnel (can also be carried out in a beaker)
3. Add 1MOL/ML Tris (PH8.0) with other solvents, mix it repeatedly and let it stand until it is layered; release the lower yellow phenol solution and discard the upper layer
4. Add about 1g/100ml of phenol in solid Tris, shake well, remove the water phase
5. Add 0.1MOL/MLTris (PH8.0) to balance several times, until PH is 8.0
6. Add 0.1MOL/MLTris (PH8.0) to the brown bottle and store at 4 degrees
7. If the yellow color disappears or becomes pink (indicating that the phenol has been oxidized), it cannot be used
Note: The re-distilled phenol is white needle-shaped crystals, which have strong corrosiveness. Avoid skin contact or inhalation. If it is found to be red or brown, it indicates that oxidation has occurred and cannot be used. Storage requirements: -20℃, protected from light. Tris-saturated phenol is a toxic and irritating substance and needs to be operated in a fume hood.
In chemiluminescence analysis, the luminescence intensity of acridine ester is influenced by various factors, such as reaction medium, temperature, time, and excitation light source energy. To achieve good detection results, it is necessary to comprehensively consider and optimize these factors. Meanwhile, attention should be paid to controlling and standardizing experimental conditions to ensure accurate and reliable results. Thoroughly studying these influencing factors will help promote the development of chemiluminescence analysis methods.