Critical Numbers for NEET
| Parameter | Value | Context |
|---|
| PCR denaturation temperature | 94-98°C | Breaks H-bonds between DNA strands |
| PCR annealing temperature | 50-65°C | Primer hybridization |
| PCR extension temperature | 72°C | Taq polymerase optimum |
| Denaturation duration | 15-30 seconds | Per cycle |
| Annealing duration | 20-40 seconds | Per cycle |
| Extension rate (Taq) | ~1 kb/minute | Use for extension time calculation |
| Heat shock temperature | 42°C | For E. coli transformation |
| Heat shock duration | 60-90 seconds | After CaCl2 treatment |
| EcoRI recognition | 5'-GAATTC-3' | 6-bp palindrome; sticky ends |
| EcoRI sticky end overhang | 5'-AATT-3' (4 nt) | After G↓AATTC cleavage |
| PCR amplification | 2^n copies | After n cycles from 1 molecule |
| 30 PCR cycles | ~10^9 (1 billion) copies | 2^30 = 1,073,741,824 |
| Restriction enzymes known | >900 | From >230 bacterial strains |
| pBR322 size | 4361 bp | Circular plasmid |
| pBR322 copy number | ~15-20 copies/cell | Determined by ColE1 ori |
| Lambda phage insert capacity | Up to ~23 kb | Larger than pBR322 (~10 kb) |
| Ethidium bromide visualization | Orange; under UV light | Intercalates between base pairs |
PCR Copy Number Formula
Final copy number = (Initial molecules) × 2^n
Where:
- n = number of PCR cycles
- Starting from 1 molecule:
- 10 cycles = 2^10 ≈ 1,000 copies
- 20 cycles = 2^20 ≈ 1,000,000 copies
- 30 cycles = 2^30 ≈ 1,000,000,000 copies
Extension Time Calculation
Extension time (minutes) = Insert size (kb) / 1 kb per minute
Examples:
- 500 bp insert: 0.5 min = 30 seconds
- 1000 bp insert: 1.0 min = 60 seconds
- 3000 bp insert: 3.0 min = 180 seconds
Restriction Fragment Analysis
For CIRCULAR DNA:
Number of cuts = Number of fragments produced
n cuts → n fragments
Verify: Sum of all fragment sizes = total plasmid size
For LINEAR DNA:
n cuts → n + 1 fragments