Extraction Calculators

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Extraction is the separation of a desired substance from a complex mixture using a difference in solubility, density, or affinity between phases. In molecular biology, nucleic acid extraction (DNA/RNA) isolates genetic material from cells for downstream analysis. In chemistry, liquid-liquid extraction (LLE) separates compounds between immiscible solvents based on partition coefficients. Extraction is also used for plant secondary metabolites, pharmaceuticals, proteins, and metals. Key principles include: solubility in the target solvent; pH effects on ionizable molecules; and multiple extraction steps to improve yield.

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DNA Extraction (CTAB or Column-Based)

Lysis: disrupt cell walls/membranes (CTAB for plants/fungi; SDS + proteinase K for animal tissue; bead beating for microbes). Protein removal: phenol-chloroform extraction or proteinase K digestion. DNA precipitation: ethanol or isopropanol + salt (Na⁺ or NH₄⁺ stabilizes DNA). Washing: 70% ethanol removes salts. Resuspension: TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) or nuclease-free water. Quality: NanoDrop A260/A280 ≈ 1.8–2.0; agarose gel for integrity.

RNA Extraction (TRIzol / Column)

TRIzol (phenol-guanidinium thiocyanate): lyses cells and denatures proteins simultaneously; add chloroform, centrifuge → aqueous phase contains RNA; precipitate with isopropanol. Column-based (RNeasy): spin column with silica membrane selectively binds RNA under high salt/ethanol conditions; elute with water. RNase-free conditions are critical: wear gloves, use DEPC-treated water or RNase-free reagents. Quality: RNA integrity number (RIN) ≥ 8 for RNA-seq; A260/A280 ≈ 2.0.

Liquid-Liquid Extraction (Chemistry)

Partition between two immiscible solvents based on Kow. D = [analyte]_organic / [analyte]_aqueous. Multiple extractions (n×small volume) > single extraction (1×large volume) for better yield: Recovery = 1 − [1/(1+D×Vorg/Vaq)]^n.

Glossary

DNA Extraction
Isolation of DNA from biological material by lysis, protein removal, and precipitation; quality assessed by A260/A280 ≈ 1.8–2.0 (NanoDrop) and Qubit fluorometry for accurate quantification.
RNA Integrity Number (RIN)
A score from 1–10 measuring RNA quality from Bioanalyzer/TapeStation; RIN ≥ 8 required for RNA-seq; based on ratio of ribosomal RNA peaks and extent of degradation products.
Liquid-Liquid Extraction (LLE)
Separation between immiscible solvents based on partition coefficient; D = [analyte]_organic/[analyte]_aqueous; multiple small-volume extractions more efficient than one large; pH-adjustable for ionizable compounds.

Frequently Asked Questions

DNA extraction proceeds through three main stages: (1) Lysis: break open cells to release contents. Animal/human tissue: SDS + proteinase K (degrades proteins); heat lysis. Plant tissue: liquid nitrogen grinding + CTAB buffer (cetyltrimethylammonium bromide — disrupts cell walls and removes polysaccharides). Bacteria: bead beating, enzymatic lysis (lysozyme), or alkaline lysis. (2) Purification: remove proteins, lipids, RNA. Phenol-chloroform: phenol denatures proteins; DNA partitions to aqueous phase. Column purification: silica membrane binds DNA in high-salt/ethanol buffer; proteins and RNA flow through; DNA eluted with low-salt buffer or water. (3) Precipitation/concentration: add ice-cold ethanol + salt → DNA precipitates; centrifuge; wash 70% ethanol; air dry; resuspend in TE or water. Quality assessment: NanoDrop (A260/A280 ≈ 1.8), Qubit (fluorometric quantification), gel electrophoresis.

RNA extraction is more challenging because: RNases are ubiquitous enzymes that rapidly degrade RNA — present on skin, dust, glassware, and in the sample itself. Unlike DNA, RNA has no complementary strand protection, making single-stranded RNA highly susceptible to RNase cleavage. Precautions: wear gloves; use RNase-free (DEPC-treated or certified) tubes, tips, and water; clean bench with RNase decontaminant (RNaseZap); work quickly on ice. mRNA is especially labile (half-life minutes in cells; degrades rapidly post-lysis without proper conditions). TRIzol denaturing conditions (phenol + guanidinium thiocyanate) immediately inactivate RNases upon cell lysis — this is key to TRIzol's effectiveness. RNA quality is assessed by RIN (RNA integrity number) from Bioanalyzer/TapeStation (RIN ≥ 8 for RNA-seq).

LLE separates compounds between two immiscible solvents (typically an organic solvent and water) based on their relative solubility in each phase. Distribution coefficient D = [analyte]_organic / [analyte]_aqueous. High D: compound prefers organic phase. Low D: compound prefers aqueous phase. pH affects ionizable compounds: weak acids (carboxylic acids, phenols) are un-ionized in acidic solution → lipophilic → extract into organic phase. In basic solution → ionized → remain in water. Strategy: adjust pH to keep analyte un-ionized for organic extraction; then back-extract by adjusting pH to ionize and shift to aqueous phase. Multiple extractions improve yield: 3 extractions with Vorg/3 each are more efficient than 1 extraction with Vorg total.

DNA quality assessment: Concentration and purity (NanoDrop): A260/A280 ≈ 1.8–2.0 for pure DNA; lower = protein/phenol contamination; A260/A230 ≈ 2.0–2.2 (low = EDTA, guanidinium, or phenol). Accurate quantification (Qubit fluorometer): Qubit uses DNA-specific dye — more accurate than NanoDrop for downstream applications. Integrity (agarose gel): high-MW band, minimal smearing → intact DNA; smearing → degradation. RNA quality assessment: Purity (NanoDrop): A260/A280 ≈ 2.0; A260/A230 ≈ 2.0–2.2. Integrity (Bioanalyzer/TapeStation): calculates RIN (1–10); RIN ≥ 8 for RNA-seq; ≥ 6 for RT-qPCR. Gel: 28S and 18S rRNA bands at approximately 2:1 intensity ratio → intact RNA.