I need to clone one gene (1.6 kb) from Gram-positive bacteria into E. coli using pET-15b vector (>5 kb). Now, I have to digest them with Fast digest ® BamHI and XhoI enzymes (Fermentas). Once, I subjected digestion for 4 hours at 37⁰C. For, pET 15b, I observed several bands. Can anyone help me, how much exact time I need to digest using Fast digest (Fermentas) BamHI and XhoI? Earlier I used normal enzymes to clone some other genes. I had no problem. But now, I got problem with time using Fast digest enzymes? when I tried the same gene with normal enzymes (not fast digest), single bands were seen. After ligation, there are several colonies and had no insert in them. Now, I had no normal enzymes with me. I had only Fast digest enzymes. Suggest me time for Fast digest enzymes?