Run the products on a gel, purify the desired fragments, and proceed with the common or compatible end restriction enzyme digestion of the destination vector. The rate-limiting step for catalysis occurs during the enzyme's open state, but with a nearly 2-fold longer duration for dATP or dTTP incorporation than for dCTP or dGTP into complementary, homopolymeric DNA templates. Simply digest the parent vector with your chosen restriction enzyme and use T4 DNA Polymerase or the DNA Polymerase I Klenow Fragment to make the end blunt. Consistent with these nanocircuit-based observations, the enzyme's closed complex forms a phosphodiester bond in a highly efficient process >99.8% of the time, with a mean duration of only 0.3 ms for all four dNTPs. Statistical analysis determined key kinetic parameters for the enzyme's open and closed conformations. Klenow Fragment (35 exo) (low concentration) EN. Removal of 3’ overhangs or fill-in of 5’ overhangs to form blunt ends. Reactions were incubated 10 minutes at 37☌. DNA Polymerase I, Large (Klenow) fragment was originally derived as a proteolytic product of E.coli DNA polymerase that retains polymerase and 3’ > 5’ exonuclease activity. An enzymatic processivity of 42 nucleotides for a template of the same length was directly observed. Dilutions of enzyme were made in a glycerol (50) containing Klenow (35 exo) storage solution and added to 50 µl reactions containing calf thymus DNA, 1x Blue Buffer, 3H-dTTP and 100 µM dNTPs. Continuous recordings of DNA polymerase processing multiple homopolymeric DNA templates extended over 600 s and through >10,000 bond-forming events. Bioconjugating single molecules of the Klenow fragment of DNA polymerase I into electronic nanocircuits allowed electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events.