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

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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: 3620

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What is the excitation and emission wavelength for rhodamine?

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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: 3227

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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?

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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: 3239

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On the Applied Biosystems® 3730 Series DNA Analyzer, what does "Disk Drive C for the EPF file is full" mean and how can I resolve it?

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The EPF file is a temporary file that can be used for troubleshooting purposes. A new file is usually generated at the start of each run. However, if the Applied Biosystems® Data Collection Software is not shut down properly, the EPF file does not close properly and the run data for subsequent runs gets written to one file. This file gets larger and larger until it fills the drive. If you get the message, go to the EPF folder (default location is the C drive, but due to the size, service may move the folder to either the D or F drive) and delete the EPF files. After deleting them, restart the system. If this folder is empty, then the drive may be full for another reason. If the full drive is your C or E drive, please work with your local IT department to determine cause and to free up space. If the full drive is D or F, these are the Oracle drives – please contact Life Technologies Technical Support for assistance.

Answer Id: 2567

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dda04f9d634145a9c68d5dfe53b21272_FAQ

My real-time PCR run stopped before it completed. What are the possible causes?

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1) Power outage and/or power fluctuation. It is recommended that your instrument be equipped with an uninterruptible power supply (UPS). Several companies manufacture such systems for scientific equipment, and they are listed in FAQ Answer ID 1935.

2) Launching other programs, setting up another run or analyzing the data while a run is in progress. The real-time data collection consumes a large amount of hardware resources. Other applications may interfere with the run and disrupt data collection causing the software to crash.

3) Hard drive fragmentation. When the SDS and other programs run, many small program files are automatically generated and deleted in the hard drive, creating blank areas between files. This can slow down your system performance by as much as 50% since programs have to access more areas of the hard drive to run. It is recommended to defragment the hard drive once a month. Please see FAQ Answer ID 2713 for more information about how to defragment a hard drive.

Answer Id: 2716

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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?

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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: 3933

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

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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: 4158

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3e3aa687770f55c704ca997c3be81634_FAQ

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

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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: 5230

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