Phenol extraction: Difference between revisions
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== Phenol extraction of nucleic acids == |
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== Nucleic Acid Extraction with Phenol == |
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[[Image:Phenol2.svg|thumb|upright|Phenol (C<sub>6</sub>H<sub>5</sub>OH) is a water-soluble compound consisting of a [[phenyl group]] (-C<sub>6</sub>H<sub>5</sub>) bonded to a [[hydroxyl group]] (-OH).]] |
[[Image:Phenol2.svg|thumb|upright|Phenol (C<sub>6</sub>H<sub>5</sub>OH) is a water-soluble compound consisting of a [[phenyl group]] (-C<sub>6</sub>H<sub>5</sub>) bonded to a [[hydroxyl group]] (-OH).]] |
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| ⚫ | Phenol extraction is a technique often used to purify raw samples of [[Nucleic acid|nucleic acids]] taken from [[Cell (biology)|cells]].<ref>{{Cite journal |last=Kirby |first=K. S. |date=1 Jul 1957 |title=A new method for the isolation of deoxyribonucleic acids: evidence on the nature of bonds between deoxyribonucleic acid and protein |journal=Biochemical Journal |volume=66 |issue=3 |pages=495–504 |doi=10.1042/bj0660495 |issn=0264-6021 |pmc=1200047 |pmid=13459887}}</ref> To obtain nucleic acid samples, the cell must be [[Lysis|lysed]] and the nucleic acids separated from all other cell materials. Phenol is a useful compound for breaking down superfluous cell materials that would otherwise contaminate the nucleic acid sample. |
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A [[mixture]] of [[tris]]-[[ethylenediaminetetraacetic acid]] (TE) and [[phenol]] is combined with an equal volume of an aqueous [[DNA]] and RNA sample. After [[Agitation (dementia)|agitation]] and [[Centrifugation|centrifugal separation]], the aqueous layer is isolated and extracted with [[diethyl ether|ether]]. The DNA is then concentrated by [[ethanol precipitation]]. |
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Phenol is [[nonpolar]] and has a higher density than water (1.07 g/cm<sup>3</sup><ref>{{Cite web |title=Phenol |url=https://www.sigmaaldrich.com/CA/en/product/sial/77608 |url-status=live |access-date=9 Jul 2022 |website=Sigma-Aldrich}}</ref> compared to water’s 1.00 g/cm<sup>3</sup>). In a water-phenol solution, [[Denaturation (biochemistry)|denatured proteins]] and other unwanted cell components will be dissolved in the phenol, while the polar nucleic acids will be dissolved in the water.<ref>{{Cite web |last=Oswald |first=Nick |date=18 Oct 2021 |orig-date=12 Feb 2008 |title=The Basics: How Phenol Extraction of DNA Works |url=https://bitesizebio.com/384/the-basics-how-phenol-extraction-works/ |url-status=live |access-date=9 Jul 2022 |website=Bite Size Bio}}</ref> The solution can then be [[Centrifuge|centrifuged]] to separate the phenol and water into distinct organic and aqueous phases. The aqueous phase, containing the purified nucleic acids, can then be extracted. |
Phenol is [[nonpolar]] and has a higher density than water (1.07 g/cm<sup>3</sup><ref>{{Cite web |title=Phenol |url=https://www.sigmaaldrich.com/CA/en/product/sial/77608 |url-status=live |access-date=9 Jul 2022 |website=Sigma-Aldrich}}</ref> compared to water’s 1.00 g/cm<sup>3</sup>). In a water-phenol solution, [[Denaturation (biochemistry)|denatured proteins]] and other unwanted cell components will be dissolved in the phenol, while the polar nucleic acids will be dissolved in the water.<ref>{{Cite web |last=Oswald |first=Nick |date=18 Oct 2021 |orig-date=12 Feb 2008 |title=The Basics: How Phenol Extraction of DNA Works |url=https://bitesizebio.com/384/the-basics-how-phenol-extraction-works/ |url-status=live |access-date=9 Jul 2022 |website=Bite Size Bio}}</ref> The solution can then be [[Centrifuge|centrifuged]] to separate the phenol and water into distinct organic and aqueous phases. The aqueous phase, containing the purified nucleic acids, can then be extracted. |
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Phenol is often used in [[Phenol-chloroform extraction|combination with chloroform]].<ref>{{Cite journal |last1=Chan |first1=P |last2=Chan |first2=D |last3=To |first3=K |last4=Yu |first4=M |last5=Cheung |first5=J |last6=Cheng |first6=A |date=May 2001 |title=Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA |journal=Journal of Clinical Pathology |volume=54 |issue=5 |pages=401–403 |doi=10.1136/jcp.54.5.401 |issn=0021-9746 |pmc=1731425 |pmid=11328843}}</ref> Adding chloroform along with phenol ensures a clear separation between the aqueous and organic phases. Chloroform and phenol are [[Miscibility|miscible]] and mix to form a denser solution than phenol alone, therefore making the organic and aqueous layers more likely to separate and form distinct phases. There is also less cross-contamination from the organic phase into the aqueous phase. This is useful when the aqueous phase is being removed from the solution to obtain a pure nucleic acid sample. |
Phenol is often used in [[Phenol-chloroform extraction|combination with chloroform]].<ref>{{Cite journal |last1=Chan |first1=P |last2=Chan |first2=D |last3=To |first3=K |last4=Yu |first4=M |last5=Cheung |first5=J |last6=Cheng |first6=A |date=May 2001 |title=Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA |journal=Journal of Clinical Pathology |volume=54 |issue=5 |pages=401–403 |doi=10.1136/jcp.54.5.401 |issn=0021-9746 |pmc=1731425 |pmid=11328843}}</ref> Adding chloroform along with phenol ensures a clear separation between the aqueous and organic phases. Chloroform and phenol are [[Miscibility|miscible]] and mix to form a denser solution than phenol alone, therefore making the organic and aqueous layers more likely to separate and form distinct phases. There is also less cross-contamination from the organic phase into the aqueous phase. This is useful when the aqueous phase is being removed from the solution to obtain a pure nucleic acid sample. |
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For phenol extraction to be effective, the pH of the solution must be adjusted according to what is being extracted. In the case of DNA |
For phenol extraction to be effective, the pH of the solution must be adjusted according to what is being extracted. In the case of DNA purification, a pH of 7.0–8.0 is used. If an experiment aims to obtain samples of purified [[RNA]], a pH of around 4.5 is used. Due to the negative charge on the backbone of DNA from the attached [[phosphate]] groups decreasing the pH of a solution will lead to neutralization. At pH 4.5, [[Hydrogen ion|hydrogen ions]] neutralize the negative charges on the phosphate groups, and cause the DNA to dissolve in the organic phase, allowing RNA to be isolated by itself in the aqueous phase. |
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==See also== |
==See also== |
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Revision as of 09:56, 13 April 2023
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Phenol extraction refers to a laboratory procedure to purify nucleic acid samples using a phenol solution.
Phenol extraction may also refer to the process of extracting and isolating phenols from raw materials, such as coal tar. These purified phenols are used in many industrial and medical compounds and are used as precursors in some synthesis reactions.
Phenol extraction of nucleic acids
Phenol extraction is a technique often used to purify raw samples of nucleic acids taken from cells.[1] To obtain nucleic acid samples, the cell must be lysed and the nucleic acids separated from all other cell materials. Phenol is a useful compound for breaking down superfluous cell materials that would otherwise contaminate the nucleic acid sample.
Phenol is nonpolar and has a higher density than water (1.07 g/cm3[2] compared to water’s 1.00 g/cm3). In a water-phenol solution, denatured proteins and other unwanted cell components will be dissolved in the phenol, while the polar nucleic acids will be dissolved in the water.[3] The solution can then be centrifuged to separate the phenol and water into distinct organic and aqueous phases. The aqueous phase, containing the purified nucleic acids, can then be extracted.
Phenol is often used in combination with chloroform.[4] Adding chloroform along with phenol ensures a clear separation between the aqueous and organic phases. Chloroform and phenol are miscible and mix to form a denser solution than phenol alone, therefore making the organic and aqueous layers more likely to separate and form distinct phases. There is also less cross-contamination from the organic phase into the aqueous phase. This is useful when the aqueous phase is being removed from the solution to obtain a pure nucleic acid sample.
For phenol extraction to be effective, the pH of the solution must be adjusted according to what is being extracted. In the case of DNA purification, a pH of 7.0–8.0 is used. If an experiment aims to obtain samples of purified RNA, a pH of around 4.5 is used. Due to the negative charge on the backbone of DNA from the attached phosphate groups decreasing the pH of a solution will lead to neutralization. At pH 4.5, hydrogen ions neutralize the negative charges on the phosphate groups, and cause the DNA to dissolve in the organic phase, allowing RNA to be isolated by itself in the aqueous phase.
See also
References
- ^ Kirby, K. S. (1 Jul 1957). "A new method for the isolation of deoxyribonucleic acids: evidence on the nature of bonds between deoxyribonucleic acid and protein". Biochemical Journal. 66 (3): 495–504. doi:10.1042/bj0660495. ISSN 0264-6021. PMC 1200047. PMID 13459887.
- ^ "Phenol". Sigma-Aldrich. Retrieved 9 Jul 2022.
{{cite web}}: CS1 maint: url-status (link) - ^ Oswald, Nick (18 Oct 2021) [12 Feb 2008]. "The Basics: How Phenol Extraction of DNA Works". Bite Size Bio. Retrieved 9 Jul 2022.
{{cite web}}: CS1 maint: url-status (link) - ^ Chan, P; Chan, D; To, K; Yu, M; Cheung, J; Cheng, A (May 2001). "Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA". Journal of Clinical Pathology. 54 (5): 401–403. doi:10.1136/jcp.54.5.401. ISSN 0021-9746. PMC 1731425. PMID 11328843.