Experimental Protocol
When programming the Experimental Protocol of your PCR, try the following:
· To optimize the PCR reaction, always titrate the MgCl2 concentration first. In general, the optimal MgCl2 concentration ranges from 1–5 mM for PCR, and 3–7 mM for RT-PCR.
· Set the annealing temperature as high as your primers allow.
· Lower the annealing time to 1–5 seconds. Caution: This strategy can only be used for reactions performed in the presence of SYBR Green I!
· Avoid over-amplification by reducing the number of cycles, e.g. to 40.
· If the melting temperatures of the desired product and the primers are known, acquire the fluorescence signal during each cycle at a temperature just below the Tm of the product and above the Tm of the primer dimers. Note: This will eliminate detection of nonspecific fluorescence.
Hot Start
To prevent nonspecific primer elongation, use the ”Hot Start“ method. In this method, at least one of the essential reaction components is separated from the reaction mixture (or blocked /inhibited) until the temperature in the tubes has exceeded the optimal primer annealing temperature.For Hot Start with the LightCycler, we recommend using commercially available anti-Taq DNA polymerase antibodies (e.g., TaqStart Antibody from Clontech [Cat. No. 5400-1, 5400-2]).This antibody keeps the polymerase inactive until the temperature rises above 70°C. The antibody is inactivated by the same heating step that denatures the target DNA.
To use the anti-Taq antibody in a LightCycler PCR, do the following:
Order of Optimization
The strategies described above will reduce primer dimers in your LightCycler PCR. Use them in the following order:
1. Reduce delays in workflow.
2. Optimize primers.
3. Increase the annealing temperature, and, if possible, reduce annealing time.
4. Use Hot Start.
5. Reduce the number of PCR cycles.
