Annealing Temperature Calculator
Calculate optimal annealing temperature for PCR reactions using primer and target melting temperatures
Calculate PCR Annealing Temperature
Melting temperature of the less stable primer
Melting temperature of the target DNA
Annealing Temperature Results
Formula used: Ta* = 0.3 × Tmp + 0.7 × Tmt - 14.9
Input temperatures: Primer: 0.0°C, Target: 0.0°C
Optimal range: PCR annealing typically occurs between 50-65°C
Temperature Analysis
Example Calculation
Cat Gene PCR Example
Target DNA: ~2 kb cat gene sequence
Target melting temperature (Tmt): 88.6°C
Primer 1: GGGGGATCTTTCTCTATAGGAAACAATTAA (Tmp = 65.5°C)
Primer 2: CACAAGCACACATGCGCACATTTGCACACA (Tmp = 74.6°C)
Less stable primer: 65.5°C (use this value)
Calculation
Ta* = 0.3 × 65.5 + 0.7 × 88.6 - 14.9
Ta* = 19.65 + 62.02 - 14.9
Ta* = 66.77°C
PCR Thermal Cycle Steps
Denaturation
94-98°C for 20-30 seconds
DNA strands separate
Annealing
50-65°C for 20-40 seconds
Primers bind to target DNA
Elongation
75-80°C
DNA polymerase extends primers
PCR Tips
Use the melting temperature of the less stable primer
Optimal annealing range is typically 50-65°C
Lower temperatures may cause non-specific binding
Higher temperatures may prevent primer binding
Understanding PCR Annealing Temperature
What is Annealing Temperature?
The annealing temperature is the temperature at which primers bind to their complementary sequences on the target DNA during PCR. It's a critical parameter that determines the specificity and efficiency of the PCR reaction.
Why is it Important?
- •Ensures specific primer binding to target sequences
- •Prevents non-specific amplification
- •Optimizes PCR reaction efficiency
- •Reduces primer-dimer formation
Formula Explanation
Ta* = 0.3 × Tmp + 0.7 × Tmt - 14.9
- Ta*: Optimal annealing temperature (°C)
- Tmp: Melting temperature of less stable primer (°C)
- Tmt: Melting temperature of target DNA (°C)
- 14.9: Empirical constant for Celsius
Note: For Fahrenheit use constant 58.82, for Kelvin use 288.05