How do you buffer phenol?

How do you buffer phenol?

Phenol extraction is frequently used to remove proteins from nucleic acid solutions. A common protocol is to add an equal volume of buffer-saturated phenol or phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v) to an aqueous nucleic acid solution, vortex, and centrifuge at 14,000 x g for 1 min to separate the phases.

How do you prepare an equilibrated phenol?


  1. Remove the crystalline phenol from the -20°C freezer and thaw it at 60-65°C.
  2. Add desired volume of phenol to an appropriately sized bottle.
  3. Add equal volume of 10X TE to the phenol.
  4. Shake vigorously and allow the layers to separate.
  5. Remove the aqueous (top) layer.

How do you increase the pH of phenol?

Extract liquid phenol using a saturating amount (s.t. 2 liquid phases form) of 1M Tris pH 8.0. This will saturate the phenol with water as well as raise the pH to the desired 8.0.

How do you mix chloroform and phenol?

  1. Prepare ml phenol:chloroform (1:1) as follows: ml phenol (pH 7.5) ml chloroform.
  2. Mix and use directly for DNA isolation.
  3. In case you want to store aliquots: add to 10-20 ml phenol/chloroform mix, 10 ml 50 mM Tris-HCl (pH 8) and freeze at -20ºC. © 2015-2022 eLabProtocols.

What is equilibrated phenol?

Phenol, also known as carbolic acid, is an organic compound with the chemical formula C6H5OH. Before use, phenol must be equilibrated to a pH of >7.8 because the DNA partitions into the organic phase at acid pH. Phenol readily undergoes oxidation and must therefore be redistilled before use.

How do you use phenol?

Phenols are widely used in household products and as intermediates for industrial synthesis. For example, phenol itself is used (in low concentrations) as a disinfectant in household cleaners and in mouthwash. Phenol may have been the first surgical antiseptic.

How do you prepare a saturated buffer phenol?

Preparation of Buffer A saturated phenol: 1. Melt an aliquot of redistilled phenol in a 65°C water bath. 2. Add 8-hydroxyquinoline to a final concentration of 0.1 % w/v to the phenol.

What does phenol chloroform do?

As we said earlier, phenol-chloroform isoamyl alcohol relies on the principle of liquid-liquid extraction of biomolecules. It denatures the protein portion of a cell and removes it followed by separating genomic DNA into a soluble phase.

How is Tris equilibrated phenol prepared?

Redistillation of phenol at 182°C under nitrogen removes oxidized products from the phenol. To prepare Tris-saturated phenol, phenol is equilibrated with Tris.Cl (pH 8.0) solution until the pH of phenol reaches ≈8.0. Often a small amount of 8-hydroxyquinoline (0.1%) is added to the phenol.

What is phenyl used for?

Phenyl is a strong deodorant and germicide for disinfecting areas covering places like hospital, nursing homes, drains, lavatory, toilets, and cowsheds and is extensively used for sanitation purpose.

What is buffer saturated phenol used for?

UltraPure™ Buffer-Saturated Phenol is used in the purification of nucleic acids. The reagent, which consists of UltraPure™ Phenol that has been saturated with Tris-HCl buffer, is already buffer equilibrated to pH >7.4.

How many μl of buffer is needed to prepare phenol?

For 10 mL of phenol, use 650 μL of buffer. For 1 mL of phenol, use 65 μL of buffer. What is the final pH of Phenol solution, Product P4557, after adding the buffer?

What is the pH of ultrapure™ buffer-saturated phenol?

The reagent, which consists of UltraPure™ Phenol that has been saturated with Tris-HCl buffer, is already buffer equilibrated to pH >7.4. When mixtures are extracted with UltraPure™ Buffer- Saturated Phenol, proteins are denatured and collect in the organic phase or at the interphase, while most nucleic acids remain in the aqueous phase.

How long does it take to equilibrate phenol?

This phenol solution (Product No. P4557) includes a separate bottle of equilibration buffer (Product No. B5658) to adjust the phenol phase to pH 7.9±0.2. After addition of the buffer to the phenol, it may take about 6 hours (mixture can be kept at 2-8 °C) until the bottom phase (phenol) becomes clear.