Should I use a three-step or a two-step PCR protocol?

Three-step PCR includes denaturation, annealing, and extension steps. This type of protocol should be used when the Tm of the primers is lower than the extension temperature or is less than 68°C. If the melting temperature of the primer (Tm) is close to the extension temperature (72°C) or a few degrees lower, consider using a two-step PCR protocol that includes a denaturation step and a combined [...]

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. [...]

Are there any special considerations when handling PCR enzymes?

To avoid contamination, gloves should be worn when handling enzyme tubes, and fingers should be kept away from the tube opening. It is imperative to use a new, clean pipette tip every time you draw from a stock tube of enzyme. When pipetting enzyme from a stock tube, place the end of the tip just far enough into the liquid to obtain the desired volume. A pipette tip should not be plunged all the [...]

How should lyophilized PCR premixes be stored?

Lyophilized PCR premixes, sealed in the original foil pouch, should be stored at room temperature (20–22°C). Any unused product should also be stored at room temperature (20–22°C), either in the original foil pouch re-sealed with desiccant, or in a desiccator.PCR [...]

How are PCR polymerases shipped, and how should they be stored and handled after receipt?

Takara Bio's PCR polymerases are shipped on dry ice. All enzymes shipped on dry ice will be frozen, but this freezing process is gradual and one freeze-thaw cycle will not reduce enzyme activity. Before opening a new tube of enzyme, spin it briefly to collect the contents at the bottom of the tube. Polymerases should be stored at –20°C in a non-frost-free freezer (an acceptable range is between [...]