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What is the optimal pH of the phenol:chloroform mixture for isolation of DNA?

Answer

Partitioning of the nucleic acids in phenol is pH dependent. At pH 7.0 or higher, both DNA and RNA partition into the aqueous phase. At an acidic pH (below 7.0) DNA is denatured and will move into the organic phase, but the RNA remains in the aqueous phase. The mixture should be adjusted to at least pH 7.4 for work with DNA.

Answer Id: E3620

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Recently I came across a DNA purification technique, which uses urea during phenol extraction. What is the purpose of using urea?

Answer

Using urea during phenol extraction denatures the protein associated with the DNA and the proteins that bind the genomic DNA to the cell wall.

Answer Id: E3239

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When using a proofreading polymerase such as Pfx, ThermalAce™, Pfu, or Vent, is it necessary to neutralize or remove the enzyme after regenerating A-overhangs with a Taq polymerase?

Answer

It is recommended to remove the enzymes via phenol-chloroform extraction to prevent the proofreading enzyme from removing the A-overhang again after Taq incubation. If not done, TA cloning efficiencies could be 4-10 fold lower. Alternatively, the PCR product can be cleaned up by gel purification or PCR cleanup column.

Answer Id: E3933

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Do you have any information on DNA and RNA purification using phenol chloroform and alcohol precipitation?

Answer

Phenol extraction of proteins:

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.

Studies at Thermo Fisher Scientific have shown that the concentration of NaCl in the aqueous solution should not exceed 0.5 M for good recovery of DNA. Residual phenol can be removed from the aqueous phase by extraction with an equal volume of chloroform or ether. After extraction, DNA is usually precipitated with ammonium acetate and ethanol as described in another protocol on this server. Ref. Karger, B. D. (1989) FOCUS™ 11, 14.

A good source of general information on the properties of phenol can be found in Wallace, Donald M. “Large and Small-Scale Phenol Extractions”. Methods in Enz. Volume 152 guide to Molecular Cloning Techniques. 1987. Academic Press, Inc. Berger and Kimmel, eds. Chap.4, pg 33-41.

(a) At pH 5 to 6 DNA is selectively retained in the organic phase and interphase, leaving RNA in the aqueous phase. Therefore a pH greater than 7 is needed if DNA is to be extracted.

(b) At pH values below 7.6, poly A+ RNA is lost to the organic phase if chloroform is not present.

(c) Optimal RNA yields in phenol extraction are obtained if the salt concentration is less than 0.15 M NaCl. Salt concentration in the sample is not a factor for larger DNA molecules.

To store RNA after extraction use DEPC-treated water.

Answer Id: E5230

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What is the recommended protocol for phenol-extraction removal of proteins from nucleic acid containing solutions?

Answer

Below is a commonly used protocol:

(1) 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.

(2) Vortex, and centrifuge at 14,000 x g for 1 min to separate the phases.

(3) The concentration of NaCl in the aqueous solution should not exceed 0.5 M for good recovery of DNA.

(4) Residual phenol can be removed from the aqueous phase by extraction with an equal volume of chloroform or ether.

(5) After extraction, DNA is usually precipitated with ammonium acetate and ethanol.

Answer Id: E4158

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