I shared it with everyone a while agoPCRProcess control of reactionLet's talk todayPCRThe operational technique. Practical operation can be said to be the wholePCRThe most easily overlooked yet crucial part of the experimental process.

Below, the editor will share some of my own experiences and insights, which only represent my personal opinion.

First, let's talk about the system configuration (separate components, non premixed liquid). Calculate the required volume of each component before configuration, and then proceed with packaging after uniform configuration. Some students may feel sorry for the reagent and prefer the single well configuration methodBut for experiments, obtaining effective results in one go is the biggest savings. When configuring the system, it is important to pay attention to the order in which each component is added: add d firstdH₂O,The remaining components are added in order of volume. When draining, insert the suction head below the liquid level to drain the liquid,After draining the liquid, it is strictly prohibited to repeatedly suck, spray, lubricate, and wash the nozzle in its original position (especially for primers, enzymes, and templates)The suction head has a certain degree of adsorption. After the liquid is discharged, if it is washed in place, the component will be sucked back into the suction head. After three to five washes, the target component may suffer serious loss. If you want to fully transfer the components inside the suction head, change the position to lubricate and wash the suction head. Attention should also be paid to the mixing of the system, which can be slowly blown with a pipette of appropriate range5-10Secondly, you can also slowly invert and mix up and down; If a vortex oscillator is needed for mixing, try to control the speed as much as possible600rpmIf the rotation speed is too fast, it will cause significant damage to the enzyme, and the difference in whether the curve of the low copy template jumps will be more obvious. System packaging topcrDuring the reaction tube, the pipette remains in placeThe operation of "suction one hit one" is not easy to generate bubbles.

Let's talk about the experimental environment. The old saying is to configure the reaction system in the ultra clean workbench and add templates in the biosafety cabinet. Inside the ultra clean table, there won't be too many problems according to conventional operations; Biosafety cabinets are a high-risk area for aerosol pollution. The aerosols in the biosafety cabinet mainly come from the discarded suction heads after sample addition. The biosafety cabinet is mainly filled with circulating air, and the residual template in the discarded suction heads can easily diffuse throughout the internal environment of the cabinet with the circulating air. Therefore, it is necessary to pack the discarded suction heads and take them out of the cabinet in a timely manner after each experiment. Especially when encountering situations where the experiment is not completed at noon and the liver continues to explode in the afternoon, it is necessary to clean the discarded gun heads in the trash can. If no one is using them, wipe them clean and then irradiate them with ultraviolet light. The table cleaning and UV disinfection and sterilization after the experiment are basic operations, but they need to be implemented in place.

Finally, let me share how to operate the double hole experiment to improve the bundling of the curve. When configuring reaction wells, you can refer to the operation of the unified configuration system, mix the templates and systems of all reaction wells, and then package them hole by hole. This can effectively reduce the risk of jumping holes. If it is low copy amplification（Ctvalue33In the future, when the curve jumps, it is not recommended to mix the template into the reaction system before packaging. At this time, it is still safer to add the template hole by hole honestly (Poisson distribution has a significant impact).

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