The following principles can be used for your reference.
1)TaqMan probe position is as close as possible to the amplification primer (amplification product 50-150bp), but not overlapping with the primer.
2)The length is generally 18-40 mer.
3)G-C content is controlled at about 40-80%.
4)Avoid the appearance of consecutive identical bases, especially to avoid GGGG or more G.
5)Avoid G at the 5' end of the primer.
6)Choose more bases C.
7)Control the annealing temperature Tm around 68-70C.

A common laboratory method is the PAGE method. Electrophoresis is carried out using a polyacrylamide gel with 7M urea, using 20% gel for primers with less than 12 bases, 16% gel for primers with 12-60 bases, and 12% gel for primers with more than 60 bases. Take 0.2-0.50D primer, dissolve it with urea saturated solution or add urea dry powder into the primer solution until saturated, and heat it to change (95C, 2min) before sampling, the purpose of adding urea is to denaturation, and increase the specific gravity of the sample, so that it is easy to add samples. 600V electrophoresis, and then peel off the gel after a certain period of time (about 2-3 hours), detect the band type under UV light with fluorescent TLC plate, no spurious bands below the main band, indicating that there is no spurious bands under the main band, which means that there is no spurious bands under the main band, and that there is no spurious bands under the main band. If there is no heterogeneous band under the main band, it means the purity is good. Sometimes, due to insufficient denaturation, there may be a band above the main band, which is a primer secondary structure band).
Additional purity testing equipment can be used to more accurately determine the purity of the primer.
HPLC (High Performance Liquid Chromatography): Gradient elution based on different adsorption of substances. Its mechanism is: when the sample into the column, solute molecules and solvent molecules on the adsorbent surface of the active center of the competitive adsorption. (At present, the main use of reversed-phase columns).
CGE (capillary electrophoresis): A type of liquid phase separation technology that uses high voltage electric field as the driving force and a capillary tube as the separation channel to realize separation based on the differences in mobility and distribution behaviors between components in the sample to achieve the effect of purity analysis. Kingsley has used it as one of the important means of primer quality control.

If the pH of the water in which you dissolve the primers is too low or if it is contaminated with bacteria or nuclease, it can degrade the primers. Inaccurate primer incorporation may also result from not thawing and mixing the liquid sufficiently when using it. It is recommended to split the primers, avoid repeated freezing and thawing, and use 10mMTrispH7.5 buffer to dissolve the primers. There is also the possibility that there is no problem with the primers, but rather that the quality of the materials used for PCR, especially the template, is not exactly the same as previously used.

The dried primer material is very loose, and before opening the cap to dissolve it is best to centrifuge for 1 minute at 3,000-4,000 rpm or tap the tube vertically upwards on a tabletop a few times to collect the primer powder into the bottom of the tube and prevent the primer from dispersing when the cap is opened.
Add deionized sterile water or 10 mMTris pH 7.5 buffer according to the calculated volume, leave at room temperature for a few minutes, mix up and down with shaking, and centrifuge to collect the solution to the bottom of the tube. The water used to dissolve the primers should generally not be distilled because some distilled water has a low pH (pH 4-5) and the primers are unstable under these conditions. Our synthesis report form gives the amount of water to be added per tube of primer diluted to a concentration of 100 uM (i.e., 100 pmopl), and you can add an appropriate amount of nuclease-free double-distilled water (pH > 6.0) or TE buffer (pH 7.5-8.0) to suit your experimental needs.

No degradation, lyophilized primers can be stored stably at room temperature for at least 2 weeks. The normal shipping time is usually 1-3 days, so the primers you receive will not degrade!

After the primers are synthesized, they undergo a series of processing and purification steps and are spin-dried into tablets. Undissolved primers are very stable and can be stored at -20°C for 2-3 years or even longer. Dissolved primers can be stored for at least half a year at -20°C avoiding repeated freezing and thawing. If the reproducibility of the experiment is required to be higher, the synthesized OD value is larger or the synthesized amount of single primer synthesis is larger, it is recommended to dilute the dissolved primer into 100M storage solution beforehand, and store it in several portions in the refrigerator at -20C. Before use, dilute the concentrated solution into a working solution (10-20uM) and carry out the experiment. Fluorescence modification primers need to be stored away from light.