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#phenol-chloroform

2 public questions tagged with this topic.

DNA & RNA isolation

Which chemical is not typically part of the phenol-chloroform DNA extraction?

Standard phenol-chloroform DNA extraction involves phenol for protein denaturation, chloroform for phase separation and lipid removal, and isoamyl alcohol, added in a 25:24:1 ratio, as an antifoaming agent that stabilizes interphase and prevents bubbling. This mixture efficiently deproteinizes the lysate. SDS (sodium dodecyl sulfate), an anionic detergent used for membrane lysis and protein solubilization, is part of the initial lysis buffer but is not a component of the organic extraction mixture itself. It is removed prior to or during phenol treatment and precipitation steps.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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DNA & RNA isolation

During phenol-chloroform extraction, DNA is found in:

Phenol-chloroform extraction separates nucleic acids based on differential solubility. Phenol denatures proteins and chloroform improves phase separation and removes lipids. When mixed with aqueous lysate and centrifuged, two phases form: lower organic phase containing phenol-chloroform and hydrophobic molecules, and upper aqueous phase containing hydrophilic nucleic acids. DNA, being highly polar due to its phosphate backbone, remains soluble in the aqueous phase, while denatured proteins partition into organic phase or precipitate at interphase. Thus pure DNA is recovered from the aqueous layer for subsequent precipitation.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details