Monarch pcr and dna cleanup kit

UniProt IDs for all proteins mentioned in this paper are in Supplementary Table 2. All primers were synthesized by Eton Bioscience (Research Triangle Park, North Carolina) and are in Supplementary Table 3. Chromosomal DNA from C. jejuni 11168 served as template to amplify fucX with OneTaq DNA polymerase (New England Biolabs [NEB]) using primers Cj0485NdeIF and Cj0485XhoIR or Cj0485XhoIRc by PCR as recommended by manufacturer’s instructions. Primers were designed to have XhoI and NdeI restriction sites for insertion into the pET28a-TEV and pET30a cloning vectors. Vectors and inserts were digested with XhoI (Promega) and NdeI (Thermo Fast Digest) following the ThermoScientific protocol for 75 min and purified using the Monarch PCR and DNA clean up kit (NEB) using the manufacturer’s protocol. The fragment amplified with the Cj0485NdeIF primer and Cj0485XhoIR primer with the stop codon intact was ligated overnight at room temperature using T4 DNA ligase as per manufacturer’s protocol into Xho/NdeI digested pET28a-TEV to create an N-terminally-His-tagged construct. The fragment with the alternative reverse primer (Cj0485XhoIRc) that disrupted the stop codon was ligated under similar conditions into pET30a to create a C-terminally-His-tagged construct. Following ligation, constructs were transformed into E. coli DH5α and confirmed by colony PCR using Taq polymerase (Invitrogen).

Primers were designed to amplify a 465bp region of cpxA containing the predicted SNP and a 442-bp region that contained the predicted SNP within the ampC promoter region (Table 2). The PCR amplification was performed using the Q5^® High-Fidelity polymerase (New England Biolabs, United States) and the final reaction contained 1× Q5^® reaction buffer, 200 μM dNTPs, 0.5 μM of the appropriate forward and reverse primers listed in Table 2, and 0.02 U/μl Q5^® polymerase in a total volume of 25 μl. The PCR samples were run using the following protocol: denaturation at 98°C for 30 s, followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 69 (cpxA SNP) or 67°C (ampC SNP) for 30 s, elongation at 72°C for 20 s, followed by a final extension of 2 min at 72°C. The PCR products were cleaned up using the Monarch^® PCR and DNA clean-up kit (New England Biolabs, United States). The PCR samples were mixed with 125 μl of the DNA Clean-Up Binding Buffer, transferred to the Clean-Up Columns and centrifuged at 11,400 × g for 1 min. The bound DNA was washed with 200 μl DNA Wash Buffer and centrifuged at 11,400 × g for 1 min, twice, followed by elution in 20 μl molecular grade water (Sigma, United Kingdom) by centrifuging at 11,400 × g for 1 min after incubating at room temperature for 2 min. All PCR products were Sanger sequenced at GeneWiz (Takely, United Kingdom).

The naive phage-displayed Helicon libraries were constructed using previously described methods^{22 (link)}. Briefly, the Peptide Display Cloning System kit from New England Biolabs was used to construct M13KE-based libraries (New England Biolabs, Ipswich, MA). Library oligonucleotides were chemically synthesized using a mix of trimer phosphoramides (Glen Research, Sterling, VA) without codons encoding cysteine, lysine, proline, or glycine, then annealed, extended, and ligated into a digested M13KE vector. All DNA products were purified using Monarch PCR and DNA cleanup kit (New England Biolabs, Ipswich, MA). The resulting library-containing phage vector was transformed into E. coli strain ER2738 (Lucigen, Middleton, WI) by electroporation and amplified by adding the post-rescue electroporated cells to a 500 mL E. coli culture at early-log phase (OD₆₀₀ = 0.01). Phage propagation, purification, and stapling were conducted as described previously^{22 (link)}.

Mycelia were collected by vacuum filtration and the total DNA isolation was performed using 50 mg of ground fungal material, as previously described [70 (link)]. The isolated DNA samples were used for PCR amplification of the ITS1-5.8S-ITS2 region of the Internal transcribed spacer (ITS) using the previously published primers, 18ITS1 [5′-GTCCCTGCCCTTTGTA-3′] and 28ITS2 [5′-CCTGGTGGTTTCTTTTCC-3′] [71 (link)].
PCR amplification reactions were performed with a KAPA Taq PCR Kit (KAPA Biosystems, Wilmington, MA, USA) in a PTC-200 Gradient Peltier Thermal Cycler (MJ Research, Waltham, MA, USA), according to the manufacturer’s instructions. The amplification protocol for the ITS region was: 3 min at 95 °C; 35 cycles of 30 s at 95 °C, 60 s at 48 °C, 2 min at 72 °C; and a final extension 5-min incubation at 72 °C. PCR amplicons were purified and cleaned using the PCR cleanup kit (NEB, Monarch PCR and DNA Cleanup Kit). All ITS amplicons were sequenced in both directions and assessed using the program SeqMan of Lasergene Suite 11 (DNASTAR Inc., Madison, WI, USA) [72 (link)]. The final sequences were deposited into GenBank. (Acc. Nos. OM993297–OM993326).

TTV was detected in human and animal sera by nested PCR using 5′ untranslated region (5′-UTR) primers NG133, NG147, NG132 and NG134 [26 (link)]. The PCR reaction mixture (50 μL) for the first and second rounds of the nested PCR was prepared using 3 μL of viral DNA, 1.0 μM of each primer and 25 μL of Go-Taq PCR Green master mix (Promega, Madison, WI, USA). A blank containing nuclease-free water instead of DNA was included in each run of PCR. The thermal cycling conditions were set to as those reported by Cancela [27 (link)]. PCR products (110 bp) were detected in 3% agarose gel and were purified for DNA sequencing using Monarch PCR and DNA Cleanup Kit (New England Biolabs, Ipswich, MA, USA), as recommended by the manufacturer.

All specific amplicons were sequenced to ensure their identity (Genewiz, Germany) and cloned using TOPO^® TA cloning^® kit (Invitrogen) with pCR^TM4-TOPO^® cloning vector, according to the manufacturer’s instructions. After cloning, plasmids were purified using NucleoSpin Plasmid kit (Marcherey-Nagel) and linearized by enzymatic digestion using Pst1 (New England Biolabs). Finally linearized plasmids were purified using Monarch PCR and DNA Cleanup Kit (New England BioLabs), and concentrations measured on spectrophotometer NanoDrop One (Thermo Scientific) in order to calculate the number of plasmid copies per microliter. Then, 10-fold serial dilutions of each plasmid were made in 10 μg/mL sonicated salmon sperm DNA (Sigma) and these dilutions were used in qPCR to determine the standard curves, to establish the amplification efficiencies and to calculate the absolute quantifications of the genes in environmental samples. Based on standard curve results, the limits of quantification (LOQ) of the assays were determined for each gene as the lowest concentration of marker within the linear range of quantification during amplification.