I PCR my inserts then digest them and my vector Pet28b with BamHI and XhoI. The digestion of the vector with each enzyme is verified on a gel. The I perform overnight ligation in a 1:5 ratio at about 9C. Ligation screen with one vector and one insert primer shows correct bands. Then I transform into DH5a and the trouble begins. The transformation yields lots of colonies, but no matter how many I screen by colony PCR with a combination of vector and insert primers or insert primers only, none of them have the insert. I've screened almost 70 colonies from each plate and no bands.... I've optimized annealing temp. already. What is going on? It's driving me crazy. Also, my advisor says there's a lot of RNA on the gel for my colony PCR, but I don't know why this would be. Could there just be so much DNA in there that it's running as a big blob near the bottom of the gel? Thanks for your help.