(1) Centrifuge dry primer powder at 13,000 rpm for 1 min at room temperature before opening the lid.

(2) Add DEPC water with 10 times nmole value of water (μL) on the tube wall, vortex to mix, and centrifuge briefly. This is the primer stock solution (100 μM), which is stored at -20°C after use.

*The primer tube is generally 3.0~6.0nmole, add ten times the value of microliters of water (30~60μL).

(3) Take a new 1.5 mL EP tube, add 380 μL of DEPC water, pipette 10 μL of the upstream and downstream primer stock solutions into the new EP tube, mix by vortexing, and centrifuge briefly. This is primer working solution (2.5μM), which can be directly used in experiments and stored at 4°C after use.