Explain gel electrophoresis

6.2.8 · Biology › Genetic Engineering & CRISPR

Core Principle

Yeh kaam kaise karta hai?

DNA negatively charged hota hai: Sugar-phosphate backbone mein phosphate groups neutral pH par negative charges carry karte hain
Electric field force create karta hai: Jab voltage apply hota hai, DNA ko positive pole ki taraf electrostatic force experience hoti hai
Gel molecular sieve ki tarah kaam karta hai: Agarose ya polyacrylamide gel mein pores hote hain; chhota DNA asaani se nikal jaata hai, bada DNA ulfajh jaata hai aur slow ho jaata hai

Step-by-Step: Yeh Kaise Kaam Karta Hai

Kya: Molten agarose gel (0.7–2% concentration) ko ek casting tray mein comb ke saath daalo Kyun: Agarose ek mesh network mein polymerize ho jaata hai. Comb samples load karne ke liye wells banata hai. Kam % = bade pores (bade DNA ke liye), zyada % = chhote pores (chhote DNA ke liye) Kaise: Agarose powder ko buffer (TAE ya TBE) mein dissolve hone tak heat karo, thoda thanda karo, daalo, solidify hone do

2. Sample Loading

Kya: DNA samples ko loading dye (glycerol + tracking dye) ke saath mix karo, wells mein pipette karo Kyun: Loading dye samples ko dense banata hai (wells mein sink karte hain) aur visible bhi (tracking dye dikhata hai run kitna aage gaya hai) Kaise: Typically 5–20 μL per well load karo; size reference ke liye ek well mein DNA ladder (molecular weight markers) zaroor daalo

3. Electrophoresis Run

Kya: Gel ko buffer (TAE/TBE) mein submerge karo, voltage (50–150 V) 30–90 min ke liye apply karo Kyun: Buffer electricity conduct karta hai aur pH maintain karta hai. DNA negative cathode se positive anode ki taraf migrate karta hai Kaise: DNA migration distance fragment size ke log ke saath linearly decrease hoti hai — yaani $d = c - k lo g (L)$

Mobility (sahi definition): $μ = \frac{v}{E} = \frac{d / t}{V / ℓ}$ jahan $μ$ electrophoretic mobility hai (cm²·V⁻¹·s⁻¹), $v = d / t$ migration velocity hai, $E = V / ℓ$ electric field hai (voltage $V$ ko inter-electrode/gel length $ℓ$ se divide karke). Mobility ko unit field per define karna (unit voltage per nahi) ise dimensionally correct banata hai aur specific apparatus size se independent bhi.

Voltage kyun matter karta hai: Zyada field = tezi se migration LEKIN zyada zyada heat → gel melting aur band distortion. Typical: 5–10 V/cm (voltage divided by gel/electrode length).

4. Visualization

Kya: Gel ko ethidium bromide (EtBr) ya safer alternatives (SYBR Green) se stain karo, UV light mein dekho Kyun: EtBr DNA bases ke beech intercalate karta hai aur UV ke neeche orange fluoresce karta hai (~302 nm par excited hone par λ ~590 nm) Kaise: Gel ko stain solution mein 15–30 min soak karo, rinse karo, UV transilluminator par photograph lo

Worked example Worked Example 1: Restriction Digest Analysis

Setup: Tumne ek plasmid (5000 bp) ko EcoRI se cut kiya, 3000 bp aur 2000 bp ke fragments expect karte hue. Electrophoresis ke baad do bands dikh rahe hain. Prediction: 2000 bp band 3000 bp band se zyada door jayega Har step kyun:

Samples load karo: Akela plasmid (control), digest, ladder
90V par 45 min run karo: ~7 cm gel ke liye standard conditions
Ladder se compare karo: Agar ladder mein 1000, 2000, 3000, 5000 bp bands hain, tumhare unknowns 2000 aur 3000 ke saath align honge
Interpret karo: Sahi cut → do bands. 5000 bp par single band → enzyme kaam nahi kiya. Teen bands → unexpected cut site

Quantification: Har band ne kitni distance travel ki measure karo, ladder ke liye $lo g (bp)$ vs. distance plot karo (standard curve, ek straight line), apne unknowns interpolate karo.

Key Variables & Optimization

Parameter	Separation par Effect	Typical Range
Agarose %	Zyada % → chhote DNA ka better resolution	0.5–2%
Field strength	Zyada → tezi LEKIN zyada heat	5–10 V/cm
Buffer	TAE (bada DNA) vs TBE (chhota DNA, better resolution)	1× concentration
Run time	Zyada lamba → zyada separation LEKIN bands diffuse ho jaate hain	30–120 min

TAE vs TBE kyun? TBE mein zyada buffering capacity hai (pH change resist karta hai) → chhote DNA (<1000 bp) ke liye sharper bands. TAE sasta hai, bade DNA (>1 kb) aur DNA recovery ke liye better hai.

Active Recall Checks

Recall Ek 12-Saal-Ke Bache Ko Explain Karo

Socho tumhare paas alag-alag sizes ke marbles ka ek bucket mixed hai. Tumhe unhe sort karna hai. Gel electrophoresis aisa hai jaise unhe ek mote sponge par daalo aur hilao. Chhote marbles sponge ke holes se jaldi nikal jaate hain aur door chale jaate hain. Bade marbles holes mein atke rehte hain aur zyada nahi chalte. Kuch der baad, light jalao aur size ke hisaab se arranged marbles ki lines dikh jaati hain. Scientists yahi DNA pieces ke saath karte hain: unhe ek jelly (gel) mein daalo, electricity on karo (DNA mein negative charge hota hai, isliye positive side ki taraf khicha jaata hai), aur chhota DNA tezi se daudta hai jabki bada DNA slow hota hai. Phir ek special glowing dye use karte hain taaki dekh sakein har DNA size kahan gaya. Yeh batata hai ki unka experiment kaam kiya ya nahi!

Mathematical Derivation: d, log(L) Mein Linear Kyun Hai

First principles se — key hai reptation (saamp ki tarah chalna) model of DNA moving in a gel.

Free-solution driving force: Free solution mein electric force $q E$ (jahan charge $q \propto L$ ) aur hydrodynamic drag dono $L$ ke saath scale karte hain, isliye mobility size-independent hoti hai — har size ka DNA saath chalega. Isliye tumhe unhe alag karne ke liye gel ki zaroorat hai.
Gel sieving via reptation: Gel mein, ek lamba DNA molecule pore network mein saamp ki tarah ek tube mein sirf ek ek karke guzarni padti hai. Ogston/reptation theory dikhata hai ki gel pores ka fraction jo ek molecule enter kar sakti hai, uski size ke saath roughly exponentially kam hoti hai: $μ (L) \approx μ_{0} e^{- β L}$ low-field, size-fractionating regime ke liye, jahan $β$ gel concentration aur pore geometry par depend karta hai.
Velocity aur distance: $v = μ E$ aur $d = v t = μ_{0} E t e^{- β L}$ ke saath. $L$ ki narrow window par jahan ek given gel ache se resolve karta hai, yeh exponential empirically aur first order mein ek aisi curve se well-approximate hoti hai jiska calibration plot migration distance vs $lo g L$ ka ek straight line hai: $d = c - k lo g L$
Empirical log-linear fit kyun kaam karta hai: Gel ki exclusion limit (bahut bada DNA, sab well ke paas atka hai) aur resolution floor (bahut chhota DNA, sab front ke paas) ke beech, response smoothly monotonic hai. $lo g L$ vs distance plot karna is working range ko linearize kar deta hai, isliye DNA ladders ek straight standard curve se padhe jaate hain — practical justification, na ki yeh claim ki reptation har jagah exactly log-linear hai.

Physical intuition: Chhota DNA pores mein se asaani se nikal jaata hai aur aage bhag jaata hai; bada DNA dheere reptate karta hai; calibration useful separation range mein $lo g L$ mein straight line ki tarah engineer ki gayi hai.

Genetic Engineering Mein Applications

CRISPR verification: Cas9 cut karne ke baad, gel run karo confirm karne ke liye ki target DNA expected sizes mein cleave hua
Cloning: Check karo ki insert vector mein ligate hua (plasmid size mein shift)
PCR genotyping: Alag-alag alleles alag band patterns dete hain
DNA fingerprinting: Restriction fragment length polymorphism (RFLP) analysis
Quality control: Sequencing se pehle DNA integrity assess karo (sharp high-MW band = achha)

Connections

Restriction Enzymes – analyze karne ke liye DNA fragments generate karte hain
PCR – gel par run karne se pehle DNA amplify karo
Southern Blotting – specific sequence detection ke liye electrophoresis ke baad ka step
DNA Structure – DNA negatively charged kyun hai (phosphate backbone)
CRISPR-Cas9 Mechanism – gel successful target cutting verify karta hai
Plasmid Vectors – gel cloning ke baad plasmid size changes check karta hai
DNA Sequencing Preparation – correct-size fragments ki gel purification

Flashcards

#flashcards/biology

Gel electrophoresis mein DNA positive electrode ki taraf migrate kyun karta hai? :: DNA mein negatively charged sugar-phosphate backbone hota hai (phosphate groups PO₄⁻ hote hain) neutral pH par, isliye yeh electrostatic force se positive anode ki taraf attract hota hai.

Gel mein DNA fragment kitni tezi se move karta hai yeh kya determine karta hai? :: Size (molecular weight) – chhote fragments bade fragments se gel pores mein tezi se navigate karte hain. Gel concentration, field strength, aur buffer se bhi affect hota hai.

Gel electrophoresis mein DNA ladder kyun use kiya jaata hai?

Ismein known sizes (molecular weight markers) ke fragments hote hain jo ek standard ki tarah serve karte hain taaki unknown fragment sizes ko migration distances compare karke estimate kiya ja sake.

Typical agarose concentration range kya hai aur ise vary kyun karte hain?

0.5–2%. Kam % (0.5–0.7%) bade DNA fragments (>5 kb) ke liye kyunki bade pores hote hain; zyada % (1.5–2%) chhote DNA (100–500 bp) ke liye kyunki chhote pores better resolution dete hain.

Agar polarity reverse kar do (samples positive end par rakh do) toh kya hota hai?

DNA galat direction mein migrate karega, turant gel se buffer mein nikal jayega, aur staining ke baad koi bands nahi dikhenge.

Gel electrophoresis mein ethidium bromide kyun use karte hain?

EtBr DNA base pairs ke beech intercalate karta hai aur UV light ke neeche fluoresce karta hai, DNA bands visible banata hai. SYBR Green jaise alternatives zyada safe hain.

Sharp bands ki jagah smeared bands kyun aate hain?

DNA overloading, degraded DNA (DNases), bahut zyada voltage se heat, purana/dried gel, ya poor buffer quality.

Migration distance aur DNA size ke beech sahi relationship kya hai?

Distance fragment size ke log ke saath LINEARLY decrease hoti hai:

d = c - k lo g L

. Yeh

1/ lo g L

ke proportional NAHI hai;

lo g L

vs distance ka standard curve ek straight line hai.

Electrophoretic mobility ki sahi definition kya hai?

μ = v / E = (d / t) / (V / ℓ)

— velocity per unit electric field, jahan field voltage ko inter-electrode length

ℓ

se divide karke aata hai. Yeh ise dimensionally consistent aur apparatus-independent banata hai.