Add following components into a microcentrifuge tube.
PCR products 0.5-4 μl (can be increased or reduced based on PCR product yield, no more than 4 μl)
pEASY ®- Blunt Zero Cloning Vector 1 μl
Gently mix well, incubate at room temperature (20oC-37oC) for 5 minutes. After reaction, place the tube on ice.
1. Optimal amount of insert
Molar ratio of vector to insert = 1:7 (1 kb, ~20 ng; 2 kb, ~40 ng)
2.Optimal volume of vector: 1 μl
3.Optimal reaction volume: 3~5 μl
4.Optimal incubation time
(1)0.1~1 kb (including 1 kb): 5~10 minutes
(2)1~2 kb (including 2 kb): 10~15 minutes
(3)2~3 kb (including 3 kb): 15~20 minutes
(4)≥3 kb: 20~30 minutes
Use the maximum incubation time if the insert is gel purified.
5.Optimal incubation temperature: for most PCR inserts, the optimal temperature is about 25oC; for some PCR inserts, optimal results can be achieved with higher temperature (up to 37oC).