| PCR reagents |
Tested range |
Optimum range |
Results of tested ranges |
| DNA concentration (μl) |
2,3,5,10,20,30,50,75 and 100 |
3 |
No amplified bands with lower concentration and presence of thinned band at higher concentration effected the repeatability |
MgCl2 (mM)
dNTPs (mM) |
1, 2, 3, 4, and 5
0.1, 0.2, 0.3 and 0.4 |
3 &
0.3 |
Excess/lower concentration increases the non specificity and yield of the product. Increased concentration reduces the free Mg2+, interfering with the enzyme. |
| Primer concentration (μM) |
1, 1.5, 2, 2.5, 3, 3.5, 4.0, 4.5 and 5.0 |
2.5 |
Lower and higher concentrations lead to absence of amplification and primer dimer formation, respectively. |
| Taq polymerase (Units) |
0.1, 0.5 and 1.0 |
1 |
Lower concentration did not show proper amplification. High concentration showed decreased specificity. |
| Initial Denaturation time interval (min) at 94°C |
2, 3, 4 and 5 |
3.0 |
Higher/lower time intervals (from optimum) leads to reduction in amplification, loss of Taq polymerase activity and lack of reproducibility |
| Annealing °C |
45, 50, 55, 60, 65 and 70 |
55 |
Higher/lower annealing temperatures (from optimum) results in difference in specificity |
| Reaction volume (μl) |
14, 16, 18 and 20 |
16 |
Influences the cost of the PCR ingredients and not effective bands obtained |
| Number of cycles |
25, 30, 35, 40, 45 and 50 |
30 |
The optimum cycles only shows effectiveness for amplification |
|
