The disease of water, as for those who drift stones, their power is also great. "- The Art of War by Sun Tzu. As long as the speed is fast enough, the stone can float on the water surface. Last month, we talked about reducing the annealing temperature change rate（Slow Optimization of PCR Reaction Program）Today, let's change the direction of optimizing PCR results and talk about how to compress the PCR reaction time.

Before discussing compression duration, it is necessary to first control the temperature of the tube/block; Introduce several concepts of normal/fast mode. The following explanation will be based on the legend of Baoheng Q9606.

Figure 1

Module temperature control:When responding to temperature changes, the real-time temperature of the module shall prevail.

Test tube temperature control:When adjusting the temperature of the reaction, the real-time temperature of the reagents inside the reaction tube shall prevail.

Figure 2

Figure 3

After the experiment editing is completed, clicking the run button will pop up a prompt box (as shown in Figure 2) to choose between fast mode or normal mode.

Taking the program in Figure 3 as an example:

Quick mode:The temperature rise and fall rate of the instrument is 8 ℃/s, indicating the full operation experiment of the temperature rise and fall rate.

Normal mode:The temperature rise and fall rate of the instrument is 4 ℃/s, indicating the half rated operation experiment of the temperature rise and fall rate.

Different application scenarios require different combinations of reaction modes. Below, we will share how to debug the reaction program of fast reagents.

Firstly, select the combination of fast mode and module temperature control for instrument operation settings to compress the reaction time. Secondly, allocate the duration of cycle segment denaturation and annealing extension reasonably while keeping the total duration unchanged. Finally, based on the screening of available reaction programs, fine adjustments are made to shorten the duration of the reaction section again or appropriately extend the duration of the reaction section under the conditions that allow for the total duration to achieve the desired reaction effect.

Because the amplification enzyme used in rapid reagents has a faster extension speed compared to enzymes in ordinary reagents, significant compression can be achieved when setting the annealing extension reaction time. Attention should be paid when compressing the denaturation reaction duration, as module temperature control is used. If the denaturation duration is too short, it will cause the actual temperature of the reagent to be less than 95 ℃ or the duration of maintaining 95 ℃ to be insufficient, resulting in insufficient cleavage of the template and affecting signal accumulation. Therefore, the duration of the denaturation reaction section should not be excessive.

The Baiheng Q9600 series can provideThe fastest speed is 8 ℃/secThe temperature rise and fall rate is high, and it has the function of equal temperature gradient (as shown in Figure 4), which not only meets the development and detection of conventional reagents, but also provides a good reaction platform for rapid reagents.

Figure 4