When optimizing PCR conditions, which conditions are particularly important?
When optimizing PCR conditions, which conditions are particularly important?
1. Initial denaturation step Preheating is sometimes required to denature complex templates (e.g., genomic DNA); 94°C for 1 min is sufficient for denaturation. Excessive heat treatment may lead to enzyme inactivation. -For Terra PCR Direct Polymerase Mix, which is used for direct PCR amplification from tissue without DNA extraction and purification, preheating at 98°C for 2 min is required. -For Takara LA Taq DNA polymerases and Advantage GC2 DNA polymerases, an initial denaturation step is required. -PrimeSTAR enzymes do not require preheating for enzyme activation. 2. Denaturing conditions Denaturing conditions should be selected by considering the thermal cycler model that will be used. A general guideline is 94–95°C for 30 sec or 98°C for 10 sec. If using a heat-resistant enzyme, such as one of the PrimeSTAR polymerases, we recommend a denaturation step of short duration and high temperature (i.e., 5–10 sec at 98°C). Denaturation at an excessively high temperature or for too long may result in loss of enzyme activity and/or damage to long templates. 3. Annealing conditions The annealing step should be adjusted for each primer set; the annealing temperature depends directly on the Tm of primers. Using annealing temperatures that are too low may result in mispriming and nonspecific amplification, leading to low yields of the desired product. Amplification efficiency and specificity can be improved by adjusting the annealing temperature according to the primer's Tm or by performing two-step PCR. -For Taq enzymes, the recommended annealing time is 30 sec. -Enzymes in the PrimeSTAR series have excellent priming efficiency. Therefore, it is important to use a short annealing time of 5–15 sec. Excessively long annealing times may lead to mispriming-induced nonspecific amplification. -When amplifying short sequences smaller than 1 kb, a three-step PCR protocol is recommended. For GC-rich targets or amplifications of long sequences (>10 kb), a two-step PCR protocol is recommended. 4. Extension step In general, an extension time of 1 min/kb is recommended. When using the high-speed enzymes SpeedSTAR HS DNA Polymerase or SapphireAmp Fast PCR Master Mix, use a reaction rate of 10 sec/kb of amplified product (i.e., 10 sec for a 1-kb product, 20 sec for a 2-kb product, etc.). PrimeSTAR Max DNA Polymerase and PrimeSTAR GXL DNA Polymerase contain a proprietary elongation factor and allow for high-speed reactions at 5–20 sec/kb. If using these enzymes with samples containing excess template, an elongation time of 1 min/kb should be used.