DNA Isolation Reviewer (HY)

DNA Isolation High Yield Reviewer (MLS 420)

THE BIG 3 DEFINITIONS

Term

One-liner

Extraction

Break cells open → release DNA

Purification

Remove contaminants (proteins, lipids)

Isolation

Extraction + Purification combined

WORKFLOW (memorize the order)

Sample → Extract → Quantify/Check Purity → Amplify (PCR) → Gel Electrophoresis → Downstream Applications

  • Purity/Quantity check = NanoDrop

  • Integrity/Size check = Gel Electrophoresis

4 BASIC ISOLATION STEPS

CSPC — Cell Lysis → Separation → Purification → Concentrate (concentrate = optional)

CELL LYSIS — HIGH YIELD CHEMICALS

Chemical

Function

CTAB / SDS

Dissolve/break membrane

β-mercaptoethanol

Breaks disulfide bonds → destroys proteins

EDTA

Inhibits nucleases

Tris (pH 8)

Maintains pH

NaCl

Stabilizes nucleic acid

ENZYME → ORGANISM MATCH

(Very commonly tested)

Enzyme

Target Cell

Proteinase K

Animal

Cellulase

Plant

Lyticase

Yeast

Lysozyme

Bacteria

MECHANICAL LYSIS METHODS

  • Mortar & pestle

  • Vortex with beads

  • Sonication — ultrasonic waves

  • French press / High-pressure homogenizer — sudden pressure change ruptures cells

5 FACTORS AFFECTING DISRUPTION

(Remember: SS-CC-P)

  1. Stability of molecules

  2. Size of sample

  3. Cohesion of cells

  4. Cell membrane type

  5. Presence of inhibitors

CENTRIFUGATION — LAYER ORDER

(Top to bottom)

  1. Aqueous phase → RNA/DNA

  2. Interphase → DNA

  3. Organic phase → proteins & lipids

Principle of centrifugation = DENSITY

PRECIPITATION & WASHING

Step

Agent Used

Precipitation

95% Ethanol (absolute) or Isopropanol + monovalent cations

Washing pellet

70–80% Ethanol

Dissolving pellet

Molecular grade water or TE buffer

Optional heating

50–55°C water bath

NUCLEIC ACID STABILIZATION

(Very high yield)

  • Store in TE buffer — neutral pH, acts as chelating agent

  • Temperature: 5°C (short-term) or -70°C (long-term)

  • AVOID freeze-thaw cycles ← commonly tested

  • Use nuclease-free, MolBio-grade plasticware

  • Aliquot samples to avoid repeated thawing

POST-ISOLATION TREATMENTS

Isolated

Treatment

DNA isolated

Apply RNase (remove RNA contamination)

RNA isolated

Apply DNase (remove DNA contamination)

ISOLATION METHODS — MASTER TABLE

Chemical-Based Methods

Method

Used For

Key Reagents

Phenol-Chloroform (GTPC)

General use

Organic solvents; produces 3-phase separation

Alkaline Extraction

Plasmid DNA from bacteria

NaOH + SDS; chromosomal DNA precipitates after neutralization

CTAB Extraction

Plant DNA

CTAB binds polysaccharides; removes contamination

EtBr-CsCl Gradient

Separating DNA forms

Ultracentrifugation; visualized via UV light; separates by density

Solid-Phase Method

4 Steps: Lysis → Binding → Washing → Elution

Uses: spin columns, magnetic beads, automated systems

Principle

Basis

Key Detail

Size exclusion (gel filtration)

Size

Large molecules elute first; small ones elute later

Ion-exchange chromatography

Charge

DNA (negative charge) binds positive matrix; eluted by changing salt or pH

Affinity chromatography

Specific binding

DNA binds silica under high salt; eluted with low salt/water

SOURCES OF NUCLEIC ACID

Microorganism

Human Samples

Virus, Bacteria, Fungi, Parasites

Blood, Saliva, Tissues, Hair, Skin, Stool, Body fluids

HIGHEST YIELD ONE-LINERS

  • EDTA → inhibits nucleases

  • NaCl → stabilizes nucleic acid

  • β-mercaptoethanol → breaks protein disulfide bonds

  • CTAB & SDS → dissolve membranes

  • Centrifugation principle → density

  • CTAB extraction → plant DNA

  • Alkaline extraction → plasmid DNA (bacteria)

  • Lysozyme → bacteria; Proteinase K → animal cells

  • NanoDrop → purity & quantity

  • Gel electrophoresis → integrity & size

  • Washing agent → 70–80% ethanol

  • Storage buffer → TE buffer

  • Never do → freeze-thaw cycles

  • DNA isolated → treat with RNase

  • RNA isolated → treat with DNase

  • Solid-phase steps → Lysis, Binding, Washing, Elution

  • Ion-exchange works by → charge interaction

  • Affinity chromatography → silica binds DNA under high salt

MEMORY TRICKS

  • CTAB = Cleans Those Aggravating Botanicals (plant DNA)

  • Lysozyme = Lyso-zyme for bacteria (lysis = bacteria association)

  • Layers after centrifuge = think blood tube: plasma (top) / buffy coat (middle) / RBCs (bottom)

  • Freeze-thaw = NO → repeated cycles degrade NA

  • Post-isolation: DNA gets RNase; RNA gets DNase (treat the opposite contamination)