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

Product FAQ

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

Was this answer helpful?

Yes
No
e923dfa7d9087492233405358c52c493_FAQ

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?

Product FAQ

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

Was this answer helpful?

Yes
No
dfc1debaf026c6d0021757924da35136_FAQ

What is the excitation and emission wavelength for rhodamine?

Product FAQ

Answer

Here are the excitation and emission values for rhodamine:
It is a mixture of X-rhodamine-5-(and-6)- isothiocyanate (5(6)-XRITC).
Emission Max is: 596 nm
Absorption Max is: 572 nm
Extinct. Coeff. is: 92,000(*)
*Note that this is for measurements of the labeling dye in methanol. The values in water/TE and attached to an oligo will be different.

This product is for Research Use Only.

Answer Id: E3227

Was this answer helpful?

Yes
No
3c72cc7ef891ff20ac547e1c3b532bec_FAQ

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

Product FAQ

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

Was this answer helpful?

Yes
No
6f04584c0fc4aa630e908f085effdf8b_FAQ

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

Product FAQ

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

Was this answer helpful?

Yes
No
a41caf43481ce4adba1a98aee400a5d8_FAQ

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

Product FAQ

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 Life Technologies™ 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

Was this answer helpful?

Yes
No
281f835f0144615df4ab32223750fcb9_FAQ