Why is temperature important in PCR?

The bacteria’s DNA polymerase is very stable at high temperatures, which means it can withstand the temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.

Why does each step of PCR require varied temperature?

The time and temperature of this step can vary depending on the nature of the template DNA and salt concentrations of buffer. Buffers with high salts (as required by some DNA polymerases) generally need higher denaturation temperatures (e.g., 98°C) to separate double-stranded DNA (Figure 3).

What three temperatures are important in PCR?

Basic PCR can be split into three general stages: denaturation, annealing and extension. Typically, a PCR protocol consists of an initial denaturation step, around 30 cycles of these three stages, a final extension step, and a holding step with a temperature of 4-10°C.

What extension temperature and time should be used for PCR and why?

During the extension step (typically 68-72°C) the polymerase extends the primer to form a nascent DNA strand. This process is repeated multiple times (typically 25-35 cycles), and because each new strand can also serve as a template for the primers, the region of interest is amplified exponentially.

What is holding temperature for PCR?

A 10°C hold will cause less wear and tear on your machine and lead to a piece of PCR equipment that runs without issues for years to come. However, for long term storage, it is best to store your completed reaction at -20°C to preserve the sample.

Why temperature of lid is always kept high as compared to temperature of reaction in PCR?

The heated lid exists to ensure that the temperature in the PCR tube is as homogeneous as possible. In the old days of PCR, when heated lids were not available yet, every 95oC cycle would cause evaporation and loss of sample, so after 25 cycles, you had nothing on the bottom of the tube.

What is melting temperature in PCR?

Primer Melting Temperature: Primer Melting Temperature (Tm) by definition is the temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. Primers with melting temperatures in the range of 52-58 oC generally produce the best results.