Ncoi hf

20 nM RNA polymerase was added to pPM872 in the presence of 200 nM Rep, UvrD, Mfd or with no helicase. Reactions were performed in replication assay conditions (40 mM HEPES pH 8; 10 mM DTT; 10 mM magnesium acetate; 2 mM ATP; 0.2 mM GTP, and UTP, 0.04 mM dNTPs; and 0.1 mg/ml BSA but without replication proteins or CTP) and incubated at 37°C for 4 minutes. A 15 μl sample was removed and cleaved with 20 units of pre-warmed NcoI-HF (NEB) at 37°C for 90 s. Immediately after the sample was removed, CTP or dH₂O was added to this reaction (200 μM final concentration CTP) and cleavage by NcoI was analysed 1, 2 and 4 minutes after addition of CTP as above. Cleavage was stopped by addition of 1 μl 0.5 M EDTA and heat inactivation (80°C for 10 minutes). Products were analysed by native agarose gel electrophoresis.

After identification of sequences of interest from the library, the corresponding genes were synthesized by Eurofins Genomics. Synthesized genes were digested with NcoI-HF and NotI-HF restriction enzymes (New England Biolabs) and ligated into the pET22b+ expression plasmid (Novagen) using T4 DNA ligase. Upon sequence confirmation, the plasmid was transformed into Rosetta(DE3) E. coli (Novagen) to facilitate protein expression.
Protein expression was performed by growth of a 50 mL culture of the above construct in Terrific Broth in the presence of 100 μg/mL ampicillin and 35 μg/mL chloramphenicol overnight at 30°C. The following day, the culture was transferred to 500 mL of fresh broth and grown for 3 h at 30°C. Protein expression was induced with IPTG at a final concentration of 0.5 mM at 30°C for 3 h. Cells were harvested by centrifugation and the protein was harvested from the periplasm by osmotic shock and purified by immobilized metal affinity chromatography and size exclusion chromatography, as described previously [20 (link), 25 (link)]. Gene sequences for Aa, Ac, Ad, and Ca through Cd are available in GenBank under accession numbers KU508539 through KU508545.

Insertion of the flap oligonucleotide abolishes an NcoI site within the λ-DNA. NcoI cleavage can thus be used to monitor the oligo insertion efficiency. After the flap oligonucleotide was ligated into the nicked λ-DNA, a Bio-Spin size exclusion column (Bio-Rad) was used to exchange the buffer into NEB Buffer 4. 1.5 μg of the λ-DNA was digested with 20 units of NcoI-HF (NEB) at 37 °C for 1 h. Digests were run on a 0.8% agarose gel containing 0.5 μg ml⁻¹ of ethidium bromide (Apex) for three hours at 100 V. Gels were imaged with a Typhoon gel imager (GE).
Alkaline agarose gels were used to monitor complete re-ligation of the nicked λ-DNA. 1.5 μg of proteinase K treated DNA was loaded into a 0.6% alkaline agarose gel using 6 × alkaline gel-loading buffer (300 mM NaOH, 6 mM EDTA, 18% (w/v) glycerol and 0.15% (w/v) Orange G (NEB)). Gels were run in 1x alkaline electrophoresis buffer (50 mM EDTA pH 8.0, 1 M NaOH) for 24 h at 20 V and 4 °C. Gels were incubated in neutralization buffer (1 M Tris-Cl pH 7.6, 1.5 M NaCl) for 45 min at RT, stained in a solution of 1 × TAE and 0.5 μg ml⁻¹ ethidium bromide for 30 min, and de-stained by soaking in ddH₂0 for 20 min. Gels were imaged with a Typhoon gel imager.