I have been doing the clonning work for more than a year now. My innitial plan is to clone the genes into pGem T easy vector and then clone into the final vector (pSNU G5H, a homemade vector). I had succesfully cloned the genes (all of them are 1.3kb) to pGem T easy vector, confirmed the sequence. However, I couldnt clone them in to the final vector. I cut both vector and the genes using MIu I restriction enzyme (I did CIAP treatment), then ligate using 2X buffer in 4 degree (also 16 degree). I didnt calculate the vector-inser vector, I just add 100 ng of vector (around 2ul), 5 ul of buffer, 1 ul of t4 ligase and the rest is the insert (my insert concentration after clean up is around 150ng/ul). There are colonies growing on my plate but when I go colony PCR, they did not show corrected band. I checked every steps but could nt find the reason why my genes doesnt go into the vector. I checked the vector after digestion and they have difference with the uncut one.