Draiii hf restriction enzyme

Making a DNA construct dual-labeled at our desired positions was an engineering challenge because a one-step PCR reaction would require an internally labeled DNA oligo to be longer than 100 bases. Such DNA oligos were not produced by IDT at the time. (Nowadays it can be purchased as an Ultramer.) To overcome this challenge, a shorter dual-labeled DNA was made by PCR, restriction digested to produce a sticky overhang, and then ligated with a biotinylated DNA fragment to form the complete construct. Briefly, perform a 2.4 mL PCR reaction using GoTaq Buffer (Promega M7921), 200 μM dNTPs, 1 μM forward primer, 1 μM reverse primer, 100 ng or less template DNA and 24 μL Taq DNA polymerase (NEB M0273L). Purify and concentrate the PCR DNA product by ethanol precipitation. Digest the DNA with DraIII-HF restriction enzyme (NEB R3510L) for 3 hr at 37°C. Purify the digested DNA product by anion exchange chromatography (Cytiva 17115301) on an FPLC instrument, followed by ethanol precipitating DNA from the peak fractions. Simultaneously prepare the biotinylated DNA fragment by annealing two single-stranded DNA oligos. Ligate the Cy5 Cy7 dual-labeled DNA with the biotinylated fragment using T4 DNA ligase (Thermo Fisher EL0011) and purify the ligated DNA by agarose gel extraction or FPLC in case of a large-scale production.