I need to clone one gene from Gram-positive bacteria into E. coli using pET-15b vector. Now, I have to digest them with Fast digest ® BamHI and XhoI enzymes (Fermentas). In Fermentas manual, it was mentioned that these Fast digest enzymes will cut the DNA in 15 min. 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 cloned with normal enzymes at 37⁰C overnight and ligated at 16⁰C overnight. I had no problems then. It got cloned. Now using Fast digest enzymes, I had some proble of digestion time I fell. Please any one help me in this regard and also suggest me ligation mix? I have transform into DH5 alpha cells.