Complete Summary and Solutions for Tools and Techniques – NCERT Class XI Biotechnology, Chapter 12 – Laboratory Methods, Analytical Techniques, Exercises

Comprehensive summary and explanation of Chapter 12 'Tools and Techniques' from the NCERT Class XI Biotechnology textbook, covering advanced techniques like microscopy, centrifugation, electrophoresis, ELISA, chromatography, spectroscopy, mass spectrometry, FISH, DNA sequencing, microarray, and flow cytometry, plus detailed answers to textbook questions and concept applications.

Updated: 1 week ago

Categories: NCERT, Class XI, Biotechnology, Chapter 12, Tools, Techniques, Laboratory Methods, Analytical Techniques, Genetics, Cell Biology, Summary, Questions, Answers

Tags: Tools, Techniques, NCERT, Class 11, Biotechnology, Microscopy, Centrifugation, Electrophoresis, ELISA, Chromatography, Spectroscopy, Mass Spectrometry, DNA Sequencing, Microarray, Flow Cytometry, Lab Methods, Chapter 12, Summary, Answers, Exercises

Tools and Techniques: Basic Concepts - Class 11 NCERT Chapter 12 - Ultimate Study Guide, Notes, Questions, Quiz 2025

Full Chapter Summary & Detailed Notes - Tools and Techniques: Basic Concepts Class 11 NCERT

Overview & Key Concepts

Chapter Goal: Understand essential lab tools and techniques in biotechnology, from basic microscopy to advanced molecular methods like DNA sequencing and flow cytometry. Exam Focus: Principles, applications, diagrams (e.g., microscope parts, gel electrophoresis setup), comparisons (e.g., types of centrifugation). 2025 Updates: Emphasis on integration with recombinant DNA tech (Unit IV), real-time applications in diagnostics/genomics. Fun Fact: Frederick Sanger's sequencing revolutionized biotech, enabling the Human Genome Project. Core Idea: These tools enable isolation, visualization, separation, and analysis of biomolecules for research/therapy. Real-World: ELISA for COVID tests; chromatography in drug purification. Ties: Links to biomolecules (Ch3), cell techniques (Ch4). Expanded: All subtopics (12.1-12.11) covered point-wise with diagram descriptions, principles, steps, and biotech relevance for visual/conceptual learning.
Wider Scope: From classical optical methods to high-tech like FISH/microarray; role in enabling experiments in genetics, immunology, and genomics.
Expanded Content: Detailed principles, types, applications; e.g., resolution in microscopy, RF in electrophoresis, Sanger sequencing steps.

Fig. 12.1: Microscope (Description)

Labelled diagram: Eyepiece, body tube, revolving nosepiece, objective lenses (4x,10x,40x,100x), arm, coarse/fine adjustment knobs, stage, condenser, sub-stage mirror, base. Visual: Compound light microscope with light path arrows.

12.1 Microscopy

Importance: Enables visualization of structures beyond naked eye; advanced forms resolve DNA/viruses.
History: Robert Hooke (1665) coined 'cell' from cork slices; Schleiden/Schwann cell theory (1838).
12.1.1 Magnification and Resolution: Magnification (M) = retinal image size with/without scope; formula M = f/(f-d) for lens. Compound M = Mo × Me (objective × eyepiece). Resolution: Smallest distance between points; key for distinguishing close objects.
Biotech Relevance: Visualizes cells/tissues for purity checks in cultures.

Fig. 12.2: Pathway of light in a light microscope (Description)

Light source → condenser lens → specimen → objective → projector lens → eyepiece → eye/magnified image. Arrows show beam path through stages.

12.1.2 Functioning of a Light Microscope

Structure: Base with stage (central hole), arm with body tube, nosepiece (objectives: 4x/10x/40x/100x), eyepiece (10x/15x), coarse/fine adjustments, condenser, light source (mirror/bulb).
Steps: Place slide on stage, align objective/eyepiece, adjust focus with knobs, illuminate via condenser/mirror.
Bright Field Microscopy: Standard; stains (carmine, eosin, safranin, methylene blue, Giemsa) for contrast.

12.1.3 Different Forms of Microscopy

Dark Field Microscopy: Oblique light beam; object glows against dark background; detects mitochondria/nuclei/vacuoles.
Phase Contrast Microscopy: Changes light phase/amplitude based on density; contrasts transparent specimens like organelles/chromosomes.
Fluorescence Microscopy: Fluorophores (acridine orange, bisbenzimide, merocyanine) emit longer wavelength light; identifies specific parts (e.g., bacteria/viruses for infection diagnosis).
Electron Microscopy: Electron beam (shorter wavelength); high resolution; vacuum operation; image on fluorescent screen. Types: Transmission (TEM: ultra-thin metal-coated sections, beam passes through); Scanning (SEM: reflected beam from gold/platinum-coated surface, 3D surface images).
Confocal Microscopy: Fluorescently labeled fixed cells/tissues; sharp high-res images of internal structures.
Applications: Biotech for 3D cell imaging, pathogen ID.

12.2 Centrifugation

Principle: Separates particles/molecules by density using centrifugal force (spinning at high rpm); gravitational force substitute.
Equipment: Centrifuge with base, rotor (holds tubes), motor, lid; cell extract spun for sedimentation.
Biotech Relevance: Isolates organelles/proteins/DNA from extracts.

Fig. 12.3: Basic structure of centrifuge (Description)

Diagram: Lid, latch, chamber, control, motor, rotor, sample tubes. Visual: Cylindrical device with spinning rotor inside.

12.2.1 Types of Centrifuge

Differential Centrifugation: Based on size/density differences; sequential speeds separate large (nuclei) to small (proteins).
Density-Gradient Centrifugation: Gradient (e.g., sucrose) in tubes; molecules band at density levels; heavier outer, lighter inner.
Ultracentrifugation: >100,000 x g for macromolecules; types: Tabletop/microfuge (low speed), high-speed, ultracentrifuge.
Applications: Isolate mitochondria/chloroplasts; virus purification.

12.3 Electrophoresis

Principle: Separates charged macromolecules (DNA/RNA/proteins) by charge-to-mass ratio in electric field; mobility ∝ charge, inversely ∝ size.
History: First observed 1807 by Strakhov/Reuss (clay particles migration).
Biotech Relevance: Analyzes PCR products, protein purity.

Fig. 12.4: Agarose gel electrophoresis unit to separate nucleic acid (Description)

Gel box with wells near negative electrode, DNA samples loaded, positive end; power on, fragments migrate by size (smaller faster); stained with ethidium bromide, visualized under UV.

12.3.1 Agarose Gel Electrophoresis

Setup: Agarose gel (polysaccharide matrix, 0.5-2% concentration) in buffer; wells for samples, DNA ladder (known sizes).
Process: Load negatively charged DNA near cathode; apply voltage; fragments move to anode (size-based: small faster through pores); visualize post-run.
Applications: Size DNA/RNA fragments; check restriction digests.

12.4 Enzyme-linked Immunosorbent Assay (ELISA)

Principle: Detects antigens/antibodies via enzyme-linked antibodies; color change quantifies presence (immunological tool).
Types: Direct (antigen coated, enzyme-Ab); Indirect (secondary enzyme-Ab); Sandwich (capture Ab, antigen, detection Ab); Competitive (competes with standard).
Steps: Coat plate with antigen, block, add sample/Ab, wash, add enzyme-substrate, measure absorbance.
Applications: HIV/pregnancy tests, protein quantification; sensitivity to pg levels.
Biotech Relevance: Diagnostics, vaccine validation.

12.5 Chromatography

Principle: Separates based on differential partitioning between mobile (liquid/gas) and stationary phases.
Types: Paper (capillary action, Rf = distance solute/distance solvent); Thin Layer (TLC: silica plate); Column (packed bed, gravity/pressure); Ion Exchange (charge-based); Affinity (specific binding, e.g., His-tag/Ni column); Gel Filtration (size-based).
Steps: Load sample on column/plate, elute with solvent, collect fractions, detect (UV/color).
Applications: Purify proteins, analyze metabolites; HPLC for high resolution.
Biotech Relevance: Recombinant protein purification.

12.6 Spectroscopy

Principle: Measures light-matter interaction; absorption/emission spectra identify/quantify molecules.
Types: UV-Vis (200-800nm, nucleic acids/proteins); IR (vibrational, functional groups); NMR (magnetic, structure); Fluorescence (excitation/emission for labeled molecules).
Steps: Prepare sample, irradiate, detect signal, analyze peaks (Beer-Lambert for concentration).
Applications: DNA purity (A260/A280=1.8), enzyme kinetics.
Biotech Relevance: Quantify biomolecules non-destructively.

12.7 Mass Spectrometry

Principle: Ionizes molecules, separates by m/z ratio, detects; gives molecular weight/sequence.
Types: MALDI-TOF (matrix-assisted laser, peptides); ESI (electrospray, proteins); GC-MS (gas chromatography coupled).
Steps: Ionize (soft/hard), accelerate, mass analyzer (quadrupole/TOF), detector; tandem MS for fragments.
Applications: Proteomics, drug identification, post-translational mods.
Biotech Relevance: High-throughput sequencing alternatives.

12.8 Fluorescence in situ Hybridisation (FISH)

Principle: Fluorescent probes hybridize to specific DNA/RNA sequences in fixed cells/tissues; visualizes location.
Steps: Fix sample, denature DNA, add labeled probe, hybridize, wash, image under fluorescence microscope.
Applications: Detect chromosomal abnormalities (e.g., aneuploidy), gene mapping, pathogen ID.
Biotech Relevance: Cytogenetics, cancer diagnostics.

12.9 DNA Sequencing

Principle: Determines nucleotide order; Sanger method (chain termination) foundational.
Sanger Steps: Denature DNA, anneal primer, add dNTPs/ddNTPs (fluorescent), extend, separate by capillary electrophoresis, read peaks.
Modern: NGS (Next-Gen: Illumina, pyrosequencing) for high-throughput.
Applications: Genome projects, mutation detection, forensics.
Biotech Relevance: Personalized medicine, variant calling.

12.10 DNA Microarray

Principle: Thousands of DNA probes on chip; hybridize with labeled sample, scan fluorescence for expression.
Types: cDNA (gene expression), SNP (variations), comparative genomic hybridization.
Steps: Spot probes, label cDNA (Cy3/Cy5), hybridize, wash, laser scan, analyze ratios.
Applications: Transcriptomics, disease profiling (cancer).
Biotech Relevance: High-throughput screening.

12.11 Flow Cytometry

Principle: Analyzes physical/chemical cell characteristics in fluid stream; laser scatters light/fluorescence.
Steps: Label cells with fluorochrome Abs, suspend in sheath fluid, hydrodynamically focus, laser interrogation, detectors (FSC/SSC/FL).
Applications: Cell sorting (FACS), apoptosis/viability, immune phenotyping.
Biotech Relevance: Single-cell analysis, stem cell research.

Summary

Tools from visualization (microscopy) to analysis (sequencing/microarray) drive biotech progress; integrate for workflows like genomics.
Interlinks: To genetic engineering (Ch13), recombinant DNA (Ch11).

Why This Guide Stands Out

Lab-focused: Step-wise protocols, visuals, applications. Free 2025 with mnemonics, disease links for retention.

Key Themes & Tips

Aspects: Resolution vs. magnification, separation principles, high-throughput vs. classical.
Tip: Memorize acronyms (TEM/SEM, ELISA types); draw setups for diagrams.

Exam Case Studies

Microscopy in cell imaging; Sanger in vaccine design.

Project & Group Ideas

Simulate gel electrophoresis with food dyes.
Debate: Classical vs. NGS sequencing costs.
Research: FISH in prenatal screening.

Key Definitions & Terms - Complete Glossary

All terms from chapter; detailed with examples, relevance. Expanded: 40+ terms grouped by subtopic; added advanced like ddNTP, fluorophore for depth/easy flashcards.

Magnification

Increases image size; M = Mo × Me. Ex: 100x objective + 10x eyepiece = 1000x. Relevance: Views tiny structures.

Resolution

Distinguishes close points; limited by wavelength. Ex: Light ~0.2μm, electron ~0.1nm. Relevance: Clear separation.

Bright Field Microscopy

Standard transmitted light with stains. Ex: Eosin for cells. Relevance: Basic cell viewing.

Dark Field Microscopy

Oblique light, glowing specimen on dark. Ex: Spirochetes. Relevance: Unstained microbes.

Phase Contrast Microscopy

Light phase shift for density contrast. Ex: Amoeba organelles. Relevance: Live cells.

Fluorescence Microscopy

Fluorophores emit light. Ex: DAPI for nuclei. Relevance: Specific labeling.

Transmission Electron Microscopy (TEM)

Beam through thin section. Ex: Virus structure. Relevance: Internal ultrastructure.

Scanning Electron Microscopy (SEM)

Surface scan with reflected electrons. Ex: Insect surface. Relevance: 3D topography.

Confocal Microscopy

Laser scans optical sections. Ex: 3D tissue. Relevance: Z-stack imaging.

Centrifugation

Density-based separation by spinning. Ex: Pellet cells. Relevance: Isolate components.

Differential Centrifugation

Size/density sequential pelleting. Ex: Nuclei at low rpm. Relevance: Organelle fractionation.

Density-Gradient Centrifugation

Band at isopycnic points. Ex: Sucrose for viruses. Relevance: Pure separation.

Ultracentrifugation

>100,000g for macromolecules. Ex: Ribosome sedimentation. Relevance: Molecular weight.

Electrophoresis

Charge-based migration in field. Ex: DNA bands. Relevance: Size analysis.

Agarose Gel Electrophoresis

Porous matrix for nucleic acids. Ex: 1% gel for 1kb DNA. Relevance: Fragment sizing.

ELISA

Enzyme-linked antigen/antibody detection. Ex: Sandwich for cytokines. Relevance: Quantification.

Chromatography

Partitioning between phases. Ex: Affinity for His-tags. Relevance: Purification.

Spectroscopy

Light interaction spectra. Ex: UV for DNA (260nm). Relevance: Concentration.

Mass Spectrometry

m/z separation of ions. Ex: MALDI for peptides. Relevance: Sequencing.

FISH

Fluorescent probe hybridization in situ. Ex: Telomere probes. Relevance: Localization.

DNA Sequencing

Nucleotide order determination. Ex: Sanger ddNTP termination. Relevance: Genomics.

DNA Microarray

Probe array for hybridization. Ex: GeneChip expression. Relevance: Transcriptome.

Flow Cytometry

Laser-based cell analysis. Ex: CD4 count. Relevance: Sorting/phenotyping.

Fluorophore

Light-emitting dye. Ex: FITC. Relevance: Labeling.

ddNTP

Dideoxynucleotide chain terminator. Ex: Sanger method. Relevance: Sequencing.

Tip: Group by technique; examples for recall. Depth: Principles tie to physics/chemistry. Errors: Confuse TEM/SEM. Historical: Hooke/Sanger. Interlinks: Ch11 rDNA. Advanced: NGS variants. Real-Life: ELISA in labs. Graphs: Spectra peaks. Coherent: Visualization → Separation → Analysis. For easy learning: Flashcard per term with diagram/app.

60+ Questions & Answers - NCERT Based (Class 11) - From Exercises & Variations

Based on chapter + expansions. Part A: 10 (1 mark, one line), Part B: 10 (4 marks, five lines), Part C: 10 (6 marks, eight lines). Answers point-wise. Easy: Structured for marks.

Part A: 1 Mark Questions (10 Qs - Short)

1. What is the magnifying power of a compound microscope calculated as?

1 Mark Answer: Product of objective and eyepiece powers (Mo × Me).

2. Name the microscopy using oblique light for dark background.

1 Mark Answer: Dark field microscopy.

3. What force separates particles in centrifugation?

1 Mark Answer: Centrifugal force based on density.

4. In electrophoresis, DNA migrates towards which electrode?

1 Mark Answer: Anode (positive).

5. What is the principle of ELISA?

1 Mark Answer: Enzyme-linked antigen-antibody detection via color change.

6. Name a chromatography type based on charge.

1 Mark Answer: Ion exchange chromatography.

7. What does UV spectroscopy measure at 260nm?

1 Mark Answer: Nucleic acid concentration.

8. In FISH, what hybridizes to target DNA?

1 Mark Answer: Fluorescent probes.

9. What terminates chain in Sanger sequencing?

1 Mark Answer: Dideoxynucleotides (ddNTPs).

10. What analyzes cells in flow cytometry?

1 Mark Answer: Laser light scatter and fluorescence.

Part B: 4 Marks Questions (10 Qs - Medium, Exactly 5 Lines Each)

1. Explain magnification and resolution in microscopy.

4 Marks Answer:

Magnification increases retinal image size; M = f/(f-d) for lens.
Compound: Mo (objective) × Me (eyepiece), e.g., 1000x total.
Resolution: Ability to separate close points; measured by min distance.
Light limited by wavelength (~0.2μm); electron higher (~0.1nm).
Relevance: High mag without res blurs; key for biotech imaging.

2. Describe functioning of light microscope.

4 Marks Answer:

Base with stage hole; arm holds body tube/nosepiece (objectives).
Eyepiece for viewing; adjustments for focus.
Light via mirror/condenser illuminates slide.
Stains (eosin/safranin) for contrast in bright field.
Path: Light → condenser → specimen → objective → eyepiece.

3. Differentiate TEM and SEM.

4 Marks Answer:

TEM: Beam through thin metal-coated section; internal 2D images.
SEM: Reflected beam from coated surface; 3D external topography.
Both use electrons for high res; vacuum, electromagnetic lenses.
TEM for ultrastructure (e.g., ribosomes); SEM for surfaces (e.g., pollen).
Apps: Virus morphology (TEM), cell interactions (SEM).

4. Explain types of centrifugation.

4 Marks Answer:

Differential: Sequential speeds for size/density (nuclei first).
Density-gradient: Bands at density levels in gradient medium.
Ultracentrifugation: High g (>100k) for molecules; includes microfuge/high-speed.
Equipment: Rotor holds tubes; rpm/time for pelleting.
Apps: Isolate mitochondria, purify ribosomes.

5. Describe agarose gel electrophoresis principle and setup.

4 Marks Answer:

Charged DNA migrates in electric field; size separates in pores.
Gel: 0.5-2% agarose in buffer; wells near cathode.
Load sample + ladder; voltage on, fragments to anode (small faster).
Visualize: Ethidium bromide stain, UV light.
Apps: Check PCR size, restriction patterns.

6. What is ELISA? Outline sandwich type steps.

4 Marks Answer:

Detects Ag/Ab via enzyme color reaction; quantitative.
Sandwich: Coat plate with capture Ab, block.
Add antigen sample, detection Ab, enzyme-Ab conjugate.
Substrate → color, absorbance measures amount.
Apps: Hormone assays, disease screening.

7. Explain ion exchange chromatography.

4 Marks Answer:

Separates by charge; stationary resin with +/− groups.
Cation: Binds +, elute with salt gradient.
Anion: Binds −, similar elution.
Steps: Load, wash unbound, elute fractions, detect UV.
Apps: Protein purification by pI.

8. Describe UV-Vis spectroscopy applications.

4 Marks Answer:

Absorption 200-800nm; Beer-Lambert for conc.
DNA/RNA: 260nm (A260=1=50μg/ml ssDNA).
Proteins: 280nm (Trp/Tyr); purity A260/280=0.6.
Steps: Blank, sample, scan spectrum.
Apps: Quantify in extracts, enzyme assays.

9. What is FISH? Give steps.

4 Marks Answer:

Localizes specific DNA/RNA in cells via fluorescent probes.
Steps: Fix/denature sample, hybridize probe overnight.
Wash unbound, counterstain, fluorescence microscope.
Colors for multiple probes (spectral karyotyping).
Apps: Detect translocations in cancer.

10. Outline Sanger DNA sequencing.

4 Marks Answer:

Chain termination with ddNTPs (no 3' OH).
Setup: Template, primer, polymerase, dNTPs + fluorescent ddNTPs.
Four reactions (A/C/G/T), extend to fragments.
Capillary gel: Size-separate, laser reads colors.
Apps: First ΦX174 genome (1977).

Part C: 6 Marks Questions (10 Qs - Long, Exactly 8 Lines Each)

1. Describe light microscope structure and light path.

6 Marks Answer:

Base supports stage with clip/hole for slide.
Arm inclines, holds body tube with nosepiece (4 objectives: 4x scanning, 10x low, 40x high, 100x oil).
Eyepiece 10x/15x; coarse/fine knobs focus.
Condenser below stage focuses light from mirror/bulb.
Path: Source → mirror → condenser → specimen → objective → body tube → eyepiece → eye.
Stains enhance contrast; total mag up to 1500x.
Res: ~0.2μm; apps: Cell morphology.
Maintenance: Clean lenses, align condenser.

2. Compare different microscopy forms with applications.

6 Marks Answer:

Bright Field: Transmitted light + stains; basic cells/tissues.
Dark Field: Oblique, unstained microbes glow; spirochetes.
Phase Contrast: Density phase shift; live organelles/amoebae.
Fluorescence: Fluorophores; specific (DAPI nuclei, FITC proteins).
TEM: Electron through section; ultra (mitochondria).
SEM: Surface electrons; 3D (pollen).
Confocal: Laser sections; 3D stacks (neurons).
Apps: Diagnostics (pathogens), research (structures).

3. Explain centrifugation types with examples.

6 Marks Answer:

Differential: Low rpm pellets large (nuclei 600g), increase for smaller (mito 10k g).
Used: Cell fractionation; seq speeds.
Density-Gradient: CsCl/sucrose gradient; bands at equilibrium (DNA 100k g).
Isopycnic: Density match, size irrelevant.
Ultracentrifugation: High speed; analytical (sed coeff), preparative.
Types: Microfuge (benchtop), high-speed refrigerated.
Safety: Balance tubes, seal rotors.
Apps: Virus isolation, protein complexes.

4. Detail agarose gel electrophoresis process and uses.

6 Marks Answer:

Prepare: Boil agarose in TAE buffer, pour comb for wells, cool.
Load: Samples + loading dye + ladder in wells near -ve.
Run: 5-10V/cm, 30-60min; DNA to +ve (neg charged).
Size: Inverse log distance; 0.8% gel for 0.5-10kb.
Stain: EtBr/SYBR, UV transilluminate, image.
Uses: PCR verification, RFLP, plasmid sizing.
Vars: PAGE for proteins, PFGE for large DNA.
Troubleshoot: Smearing = degradation, no bands = no load.

5. Describe ELISA types and sandwich protocol.

6 Marks Answer:

Direct: Ag coat, enzyme-Ab bind, substrate.
Indirect: Secondary enzyme-Ab amplifies signal.
Sandwich: Capture Ab coat, add Ag, detection Ab, enzyme-Ab.
Competitive: Sample competes with coated Ag for Ab.
Protocol: 96-well plate, 4°C overnight coat, PBS block 1h.
Incubate sample 2h, wash, add HRP-Ab 1h, TMB substrate, stop, 450nm read.
Quant: Standard curve, LOD ~ng/ml.
Apps: ELISA kits for allergens, cytokines.

6. Explain chromatography principles with affinity example.

6 Marks Answer:

Mobile phase carries, stationary retains differentially.
Adsorption: Surface binding (TLC silica).
Partition: Solubility (paper water vs. solvent).
Ion Exchange: Charge (DEAE for anions).
Gel Filtration: Size exclusion (Sephadex pores).
Affinity: Specific ligand (Ni for His-tag proteins).
Steps: Equilibrate column, load lysate, wash, elute imidazole gradient, SDS-PAGE check.
Apps: mAb purification, metabolite profiling.

7. Discuss spectroscopy types and DNA quantification.

6 Marks Answer:

UV-Vis: Electronic transitions; cuvette, monochromator.
IR: Vibrational; fingerprint spectra.
Fluorescence: Excitation/emission; sensitive for labels.
NMR: Nuclear spin; 3D structure.
DNA: A260=1 (50μg/ml ds, 33 ss); pure if A260/280=1.8, A260/230=2.0.
Steps: Blank buffer, dilute sample 1:50, read absorbance.
Contam: Phenol low 280, salt low 230.
Apps: Bradford for protein (595nm).

8. Outline mass spectrometry components and proteomics use.

6 Marks Answer:

Ionization: ESI soft for proteins, MALDI laser matrix.
Analyzer: Quadrupole filters m/z, TOF time-of-flight.
Detector: Electron multiplier.
Tandem (MS/MS): Fragment ions for sequence.
Proteomics: Digest proteins, LC-MS, peptide ID by database.
Steps: Sample prep, ionize, separate, ID peaks (mass = charge × m/z).
Res: ppm accuracy; post-trans mods.
Apps: Biomarker discovery.

9. Explain DNA microarray for gene expression.

6 Marks Answer:

Glass slide with spotted/printed probes (oligos/ cDNA).
Label mRNA (Cy3 control, Cy5 test) reverse transcribe.
Hybridize overnight, wash, laser scan ratios.
Red/green: Up/down regulated; yellow equal.
Analysis: Scatter plot, fold change >2.
Steps: Extract RNA, label, array, normalize data.
Vars: Oligo arrays for SNPs.
Apps: Cancer subtypes, drug response.

10. Describe flow cytometry principle and cell sorting.

6 Marks Answer:

Hydrodynamic focusing: Cells in stream past laser.
Scatter: FSC size, SSC granularity.
Fluorescence: Channels (FL1 FITC, FL2 PE) for markers.
Detectors: PMT amplify signals, histogram/dot plots.
Sorting (FACS): Electrostatic deflection of droplets.
Steps: Stain Abs, run 10k events/sec, gate populations.
Apps: CD34 stem cells, apoptosis (Annexin V).
Multicolor: 20+ parameters.

Tip: Diagrams for setups; practice steps. Additional 30 Qs: Variations on NGS, HPLC.

Key Concepts - In-Depth Exploration

Core ideas with examples, pitfalls, interlinks. Expanded: All 12.1-12.11 with steps/examples/pitfalls for easy learning. Depth: Calculations (e.g., A260), troubleshooting.

Magnification vs. Resolution

Steps: 1. Calc M=10x100=1000x, 2. Res=λ/2NA (Abbe). Ex: Electron 100k x clear viruses. Pitfall: High mag empty res. Interlink: Limits in biotech imaging. Depth: Oil immersion NA=1.4 boosts res.

Bright Field Microscopy

Steps: 1. Stain slide, 2. Focus 40x, 3. Observe. Ex: Blood smear Giemsa. Pitfall: Overstain quenches. Interlink: Prep for EM. Depth: Kohler illumination even field.

Fluorescence Microscopy

Steps: 1. Label fluorophore, 2. Excite 488nm, 3. Emit 520nm filter. Ex: GFP fusion proteins. Pitfall: Photobleaching. Interlink: FISH/confocal. Depth: FRET energy transfer.

Electron Microscopy (TEM/SEM)

Steps: 1. Fix/dehydrate/embed ultrathin, 2. Stain uranyl, 3. Beam/image. Ex: HIV particles. Pitfall: Artifacts from prep. Interlink: Cryo-EM modern. Depth: 1e6 x res.

Differential Centrifugation

Steps: 1. Homogenize tissue, 2. 1000g nuclei, 10k g mito, 100k g microsomes. Ex: Liver fractions. Pitfall: Overlap sizes. Interlink: Gradient refine. Depth: Svedberg units.

Agarose Gel Electrophoresis

Steps: 1. 1% gel, 2. Load 5μl, 100V 45min, 3. Image. Ex: 500bp band. Pitfall: Run too far smears. Interlink: Southern blot. Depth: Log MW vs. Rf linear.

ELISA (Sandwich)

Steps: 1. Coat Ab 1μg/ml, 2. Ag 100μl, 3. Biotin Ab, strep-HRP, TMB. Ex: IL-6 ng/ml. Pitfall: Cross-react. Interlink: Western. Depth: EC50 curve fit.

Chromatography (Affinity)

Steps: 1. Bind Ni resin, 2. Load lysate, 3. Wash 20mM imidazole, 4. Elute 250mM. Ex: GST fusion. Pitfall: Non-specific. Interlink: FPLC scale-up. Depth: Kd binding constant.

Spectroscopy (UV-Vis)

Steps: 1. λmax scan, 2. A=εcl (ε=6600 for Tyr). Ex: Protein 1mg/ml A280=1.4. Pitfall: Turbidity scatter. Interlink: Fluoro. Depth: Molar extinction coeff.

Mass Spectrometry

Steps: 1. ESI ionize, 2. TOF separate, 3. MS/MS CID fragments. Ex: Peptide mass fingerprint. Pitfall: Adducts. Interlink: Proteomics. Depth: Isotope patterns.

FISH

Steps: 1. Probe design 50-100nt, 2. 37°C hybrid 16h, 3. Counter DAPI. Ex: HER2 amp cancer. Pitfall: Off-target. Interlink: Microarray. Depth: Spectral imaging multi-color.

Sanger Sequencing

Steps: 1. PCR amplify, 2. Cycle seq dNTP/ddNTP, 3. Cap gel read. Ex: 500bp read. Pitfall: Compression G-C. Interlink: NGS. Depth: BigDye terminators.

DNA Microarray

Steps: 1. Spot 60mer probes, 2. Hybrid Cy-dye cDNA, 3. Ratio log2 FC. Ex: Yeast stress genes. Pitfall: Cross-hybrid. Interlink: RNA-seq. Depth: Affymetrix vs. Agilent.

Flow Cytometry

Steps: 1. Ab conjugate PE, 2. 488nm laser, 3. Gate live singlets. Ex: 95% CD3 T-cells. Pitfall: Comp overlap. Interlink: Sorting. Depth: Compensation matrix.

Advanced: Res calc, RF= distance migrated. Pitfalls: Contam in gels. Interlinks: Ch13 apps. Real: NGS in COVID. Depth: 12 techniques details. Examples: Sanger ΦX174. Graphs: Spectra/elution. Errors: Electrode mixup. Tips: Steps for protocols; compare tables.

Historical Perspectives - Detailed Guide

Timeline of tool development; expanded with points; links to scientists/experiments. Added Hooke, Sanger, modern NGS.

Early Microscopy (17th C)

1665: Hooke simple scope, coins 'cell' from cork.
1838: Schleiden/Schwann cell theory via observations.

Depth: Van Leeuwenhoek microbes 1670s.

Electrophoresis (19th-20th C)

1807: Strakhov/Reuss clay migration.
1930s: Tiselius proteins; Nobel 1948.

Depth: Agarose 1950s for DNA.

Centrifugation (20th C)

1920s: Svedberg ultracentrifuge; Nobel 1926.
1940s: Density gradients for viruses.

Depth: Beckman instruments 1947.

Immunoassays (1950s-70s)

1959: Yalow radioimmunoassay; Nobel 1977.
1971: ELISA by Engvall/Pearson.

Depth: Non-radio alternatives.

Chromatography/Spectroscopy (1900s)

1903: Tswett column chrom; 1913: UV-Vis.
1940s: Paper/TLC; 1950s: NMR.

Depth: HPLC 1960s.

Mass Spec & Sequencing (1970s-80s)

1977: Sanger ΦX174 seq; Nobel 1980.
1980s: MALDI/ESI MS.

Depth: Human Genome 2003.

Modern (1990s+)

1991: FISH chrom mapping.
1995: Microarray; 2000s NGS (454/Illumina).
1969: Flow cytometry; FACS 1970s.

Depth: CRISPR integration 2010s.

Tip: Link to Nobel (Sanger, Yalow). Depth: Bateson linkage ties. Examples: 1977 first genome. Graphs: Timeline milestones. Advanced: Post-HGP tools. Easy: Chrono bullets impacts.

Solved Examples - From Text with Simple Explanations

Expanded with protocols, calcs; focus on setups, troubleshooting. Added gel analysis, sequencing read.

Example 1: Microscope Magnification (Fig 12.1)

Simple Explanation: Total power for clear view.

Step 1: Select 40x objective, 10x eyepiece.
Step 2: M = 40 × 10 = 400x.
Step 3: Oil for 100x = 1000x.
Step 4: Res limits detail.
Simple Way: Objective close power × eye far power.

Example 2: Gel Electrophoresis Band Sizing

Simple Explanation: Measure distances for kb.

Step 1: Ladder: 1kb=5cm, 2kb=4cm, 5kb=2.5cm.
Step 2: Unknown band 3.5cm.
Step 3: Log MW vs. cm linear; interpolate ~1.5kb.
Step 4: Confirm with standard curve.
Simple Way: Smaller runs farther, plot log.

Example 3: ELISA OD Reading

Simple Explanation: Color to concentration.

Step 1: Standards: 0ng=0.1 OD, 10ng=0.5, 100ng=1.2.
Step 2: Sample OD=0.8.
Step 3: Linear fit, ~50ng/ml.
Step 4: Dilution factor ×10.
Simple Way: Darker color = more analyte.

Example 4: UV DNA Quant

Simple Explanation: Absorbance to μg.

Step 1: A260=0.5, dilute 1:20.
Step 2: Actual A260=10.
Step 3: 10 × 50μg/ml = 500μg/ml.
Step 4: Purity A260/280=1.9 good.
Simple Way: 1 unit = 50μg dsDNA.

Example 5: Sanger Read Interpretation

Simple Explanation: Peaks to sequence.

Step 1: Chromatogram: G peak1, A2, T3, C4.
Step 2: Read 5' GAT C... from short to long.
Step 3: Quality score >Q20.
Step 4: Assemble contigs.
Simple Way: Color peaks = bases order.

Example 6: Flow Cytometry Gating

Simple Explanation: Sort populations.

Step 1: FSC/SSC plot: Gate lymphocytes.
Step 2: FL1 (CD3) histogram: 80% positive.
Step 3: Dot plot CD4 vs CD8: 60% helper.
Step 4: Sort high FL into tube.
Step 5: Verify purity 95%.
Simple Way: Scatter sizes, color markers.

Tip: Calc practice; troubleshoot (e.g., no bands=voltage off). Added for FISH (signal count), MS (m/z).

Quick Revision Notes & Mnemonics

Concise for 12.1-12.11; mnemonics. Covers principles/steps/diffs. Expanded all subtopics.

12.1 Microscopy

Mag M=MoMe; Res min dist ( "MR Scope Limit" - MRSL).
Bright: Stains; Dark: Oblique; Phase: Density; Fluoro: Emit ( "BDFP Emit" - BDFPE).
TEM thru, SEM surf; Confocal 3D ( "TSS C3D" - TSSC).

12.2 Centrifugation

Diff size seq; Density band; Ultra high g ( "DDU Spin" - DDUS).
Rpm pellet; Microfuge low ( "RM Low" - RML).

12.3 Electrophoresis

Charge mass field; DNA -ve anode ( "CMF Anode" - CMFA).
Agarose pores size; Ladder ref ( "APS L" - APSL).

12.4 ELISA

Enz Ab color; Sandwich capture detect ( "EAC S" - EACS).
Direct simple, Indirect amp ( "DI Amp" - DIA).

12.5 Chromatography

Mobile stat partition; Ion charge, Affinity specific ( "MSP IAS" - MSPIS).
Gel size, Paper Rf ( "GP R" - GPR).

12.6 Spectroscopy

UV DNA 260; IR vib, NMR spin ( "UIRN Light" - UIRNL).
Beer A=εcl ( "BAE C" - BAEC).

12.7 Mass Spectrometry

Ion m/z; ESI soft, MALDI laser ( "IM ESML" - IMESM).
MS/MS frag seq ( "MMFS" - MMFS).

12.8 FISH

Fluo probe hybrid situ; Denat wash image ( "FPH DWI" - FPHD).
Chrom abnor ( "CA FISH" - CAF).

12.9 DNA Sequencing

Sanger ddNTP term; Cycle extend read ( "SDTCER" - SDTCE).
NGS high thru ( "NGST" - NGST).

12.10 DNA Microarray

Probe chip hybrid; Cy ratio fold ( "PCHF R" - PCHFR).
Expr profile ( "EP Array" - EPA).

12.11 Flow Cytometry

Laser scatter fluo; FSC size SSC gran ( "LSSF SG" - LSSFSG).
Gate sort ( "GS Flow" - GSF).

Overall Mnemonic: "Micro Cent Electro ELISA Chrom Spec Mass FISH Seq Micro Flow" (MCE ECM SMF SMF). Flashcards: One per subtopic. Easy: Bullets, bold keys; steps acronyms.

Key Terms & Processes - All Key

Expanded table 40+ rows; quick ref. Added advanced (e.g., chiasma no, but rf, NA).

Term/Process	Description	Example	Usage
Magnification	Image enlargement	1000x total	View details
Resolution	Point separation	0.2μm light	Clarity
Bright Field	Stained transmitted light	Cell slides	Basic imaging
Dark Field	Oblique glow	Bacteria	Unstained
Phase Contrast	Density phase shift	Live cells	Organelles
Fluorescence	Emitting dyes	GFP	Specific label
TEM	Through section electrons	Virus internal	Ultrastructure
SEM	Surface reflected	3D pollen	Topography
Confocal	Optical sections	Tissue stacks	3D recon
Centrifugation	Density spin sep	Cell pellet	Isolation
Differential	Size seq speeds	Nuclei 1000g	Fractionate
Density-Gradient	Band equilibrium	DNA CsCl	Purify
Ultracentrifuge	High g molecules	Ribosomes	Sed coeff
Electrophoresis	Charge migration	DNA bands	Size sort
Agarose Gel	Pore matrix DNA	1kb ladder	Fragment ID
ELISA	Enz immuno detect	HIV test	Quant Ag/Ab
Sandwich ELISA	Capture + detect Ab	Cytokine	Specificity
Chromatography	Phase partition	Protein column	Purify
Ion Exchange	Charge resin	DEAE anion	pI sep
Affinity	Ligand specific	His-Ni	Tag purify
Spectroscopy	Light-matter interact	UV DNA	Conc spectra
UV-Vis	Absorbance 260nm	A260=1.8 pure	Nuc quant
Mass Spec	m/z ions	Peptide MS	Mol wt seq
ESI	Electrospray ion	Proteins	Soft ionize
FISH	Probe hybrid in situ	Chrom del	Locate genes
DNA Sequencing	Nuc order	Sanger 500bp	Genome assem
ddNTP	Chain terminator	G peak	Termination
Microarray	Probe array hybrid	Gene expr	Profile transcrip
Flow Cytometry	Laser cell stream	CD4 count	Phenotype
FSC	Forward scatter size	Lymph gate	Cell vol
Fluorophore	Emitting molecule	FITC 488/520	Label detect
Rf Value	Solute/solvent dist	0.5 spot	Chrom ID
Beer-Lambert	A=εcl law	Conc calc	Quant
Chiasma	No, but CO site	Meiosis	Recomb
NA	Numerical aperture	1.4 oil	Res boost
Svedberg	Sed coeff unit	80S ribosome	Mol size
Ethidium Bromide	DNA intercalator	Gel stain	UV fluores
HRP	Horseradish peroxidase	ELISA enz	Color sub
Imidazole	Elution buffer	Affinity wash	Compete bind
TMB	Tetramethylbenzidine	ELISA sub	Blue color
Q20	Phred quality score	Seq accuracy	Base call
Cy3/Cy5	Dye labels	Microarray	Ratio green/red

Tip: Examples memory; sort technique. Easy: Table scan. Added 20 rows depth (e.g., TMB, Q20).

Key Processes & Diagrams - Solved Step-by-Step

Expanded all major; desc for diags; steps visual. Added electrophoresis run, sequencing cycle.

Process 1: Light Microscope Use (Fig 12.2)

Step-by-Step:

Step 1: Place stained slide on stage, secure clips.
Step 2: Rotate 10x objective, lower with coarse.
Step 3: Adjust light/condenser, fine focus.
Step 4: Switch 40x/100x, recenter.
Step 5: Observe/draw; clean after.
Diagram Desc: Light path arrows from bulb to eye.

Process 2: Agarose Gel Electrophoresis (Fig 12.4)

Step-by-Step:

Step 1: Dissolve 1g agarose in 100ml TAE, microwave, add EtBr.
Step 2: Pour in tray with comb, solidify 30min.
Step 3: Submerge in buffer tank, load 10μl sample.
Step 4: Run 80V 1h, disassemble.
Step 5: UV image, measure bands.
Diagram Desc: Box with gel slab, electrodes, wells, migration arrows.

Process 3: Centrifugation Fractionation

Step-by-Step:

Step 1: Homogenize tissue in buffer, filter debris.

Step 2: 800g 10min pellet nuclei.

Step 3: Sup 10k g 20min mito.

Step 4: Sup 100k g 1h microsomes.

Step 5: Resuspend, assay markers (DNA mito).

Diagram Desc: Tubes layered post-spin, pellets/sup.

Process 4: Sandwich ELISA

Step-by-Step:

Step 1: Coat wells Ab 100ng, 4°C o/n.
Step 2: Block BSA 3% 1h RT.
Step 3: Add sample 100μl 2h.
Step 4: Detection Ab 1h, HRP-Ab 1h.
Step 5: Wash PBS-T, TMB 10min, stop H2SO4, read 450nm.
Diagram Desc: Plate wells sequential layers Ab-Ag-Enz-Sub.

Process 5: Affinity Chromatography

Step-by-Step:

Step 1: Pack Ni column, equilibrate buffer pH8.
Step 2: Load clarified lysate 1ml/min.
Step 3: Wash 10CV 20mM imidazole.
Step 4: Elute gradient 50-500mM, collect 1ml fractions.
Step 5: Dialyze, SDS-PAGE Coomassie.
Diagram Desc: Column flow: Load → Wash → Elute peaks.

Process 6: Sanger Sequencing Cycle

Step-by-Step:

Step 1: Denature 95°C template/primer.
Step 2: Anneal 50°C primer.
Step 3: Extend 72°C Taq + dNTP/ddNTP mix.
Step 4: 25-30 cycles, purify products.
Step 5: Capillary run, detect colors A/G/C/T.
Diagram Desc: Thermal cycler profile, electropherogram peaks.

Process 7: Flow Cytometry Analysis

Step-by-Step:

Step 1: Incubate cells Ab 30min ice.
Step 2: Wash, resuspend 1e6/ml.
Step 3: Run flow, acquire 10k events.
Step 4: Software gate FSC/SSC, FL histograms.
Step 5: Stats % pos, MFI.
Diagram Desc: Dot plot quadrants, histogram peaks.

Tip: Draw flows; label parts. Easy: Numbered with analogies (gel as sieve).

This article has been published in CBSE Class 11 Annual Assessment. Explore everything related to it here.

Group Discussions

No forum posts available.

Easily Share with Your Tribe