Previously we used to clone HIV-1 full-length gp160 in our lab using a pcDNA Directional TOPO Expression cloning kit regularly for high throughput cloning. However, we have recently observed that this kit's efficiency has drastically reduced with absolutely nothing in colony PCR. As an alternate, we are also trying the traditional Restriction Digestion cloning method, Overlapping PCR, and NEB HiFi Assembly (Gibson Assembly) method. However, success rates are very less. What could be the possible reason for this and if anybody can suggest any good high-throughput method? Will be grateful.