Hello everybody!!! I am facing a problem regarding 3 way ligation. I am trying to ligate 2 inserts into a vector with desired gene. Every time after ligation and colonies screening i am getting the same pattern of bands which lack the one insert size. I checked and confirmed the restriction digestion of each fragment and optimized the other factors like transformation, digestion and ligation. Vector size is around 6kb and insert 1 is 500bp, insert 2 is 700bp. After restriction digestion with the appropriate enzymes i am getting the band size of 6kb and 500bp, which shows lack of 700bp (desired band size should be 6kb and 1.2kb). Experts here kindly lend their valuable suggestion. I would appreciate their comments over my problem. Thank you