Q5 hot start high fidelity dna polymerase

The coding DNA sequence (CDS) of the human PNKP (gene accession# NM_007254.4) was amplified using the Q5 hot start high fidelity DNA polymerase (New England Biolabs), with HEK293 genomic DNA as a template, and the amplified PCR fragment was cloned in pCDNA3 (containing an N-terminal FLAG tag) using HindIII–BamHI sites as described earlier (20 (link)). K142 and K226 were mutated to Arginine (K142R & K226R) or glutamine (K142Q & K226Q), individually, using the Q5 site-directed mutagenesis kit (New England Biolabs), according to the manufacturer's protocol. PCR primers (Integrated DNA Technologies, Inc.) for site-directed mutagenesis were designed using NEBaseChanger. The primers to introduce these mutations are listed in Table 2. A double mutant (K142R/K226R) was generated using the single mutant K142R as a template and then the K226R mutation was introduced in it. Similarly, a K142Q/K226Q double mutant was generated using K142Q as a template.

Libraries were prepared for sequencing as described previously with some modifications as outlined in Supplement B (24 (link), 56 (link)). Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen). Genomic DNA extracts were digested with KflI to eliminate sequence reads from integers and subsequently purified using a DNA clean and concentrator-5 kit (Zymo Research). A total of 100 ng of the genomic was linearly amplified using a dual priming oligonucleotide (268811662) and Q5 hot start high-fidelity DNA polymerase (New England Biolabs). Fifty PCR cycles were conducted with a 10-s extension in 50-μL reaction mixtures. Linear PCR products were purified as described above, c-tailed on the 3′ end, and purified. The linear c-tailed products were exponentially amplified using Q5 polymerase and the P5-Himar1-Bx (26881166x) and P716G p (273791651) primers (Table S5) for 22 to 25 PCR cycles in 25-μL reactions. The amplified libraries were fractionated on an agarose gel and stained with GelRed, and gel slices corresponding to 300 to 500 bp size were excised for gel purification. The libraries were sequenced on Hiseq X 150 PE.

Viral RNA was reverse transcribed utilizing the ProtoScript^®II First Strand cDNA Synthesis Kit (New England Biolabs GmbH, Frankfurt, Germany) and random primers according to the manufacturer’s protocol. LCMV-WE and LCMV-Docile cDNA was amplified with Taq DNA Polymerase (Qiagen, Hilden, Germany) and overlapping LCMV-specific primer pairs to cover the S-segment and L-segment of both LCMV strains. Purified PCR products (Qia Quick PCR purification Kit) were sequenced utilizing the Big Dye^TM Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems^TM) and analyzed on a Prism Genetic Analyzer 3130-16 (Applied Biosystems^TM). Terminal Sequences (5’ and 3’ ends) of S- and L-segments were obtained from viral RNA utilizing the Superscript^TM IV First-Strand Synthesis System (Thermo Fisher Scientific) according to the manufacturer’s protocol and gene specific primer for first-strand synthesis, followed by dA tailing and second strand synthesis with oligo-T-tailed primers. The second strand synthesis product was PCR amplified utilizing Q5^® Hot Start High-Fidelity DNA polymerase (New England Biolabs GmbH, Frankfurt, Germany) oligo -T-tailed primers and nested gene specific primers. Purified PCR products were sequenced with nested gene specific primers as described above.

The whole genome of SARS-CoV-2 from the clinical samples was sequenced using the Oxford Nanopore sequencing technology (Oxford Nanopore Technologies, Cambridge, United Kingdom) following the ARTIC network protocol. The SARS-CoV-2 positive RNA extracts were reverse-transcripted with LunaScript^™ RT SuperMix Kit (New England Biolabs, Ipswich, United States). Multiplex PCR with ARTIC Network V3 primer pools, tilling the complete SARS-CoV-2 genome, was performed on cDNA using Q5 Hot Start High-Fidelity DNA polymerase (New England Biolabs, Ipswich, United States). The amplicons were cleaned up with AMPure XP beads (Beckman Coulter Diagnostics, California, United States), and libraries were prepared using the ligation sequencing kit (SQK-LSK109) from Oxford Nanopore Technologies (Oxford, United Kingdom). Then, libraries were quantified using QUBIT 1X dsDNA HS Assay Kit (Invitrogen, Waltham, United States), and 15 ng of each prepared library was loaded into Oxford Nanopore MinION SpotON Flow Cells FLO-MIN106D, R9.4.1 (Oxford Nanopore Technologies, Oxford, United Kingdom (Bull et al., 2020 (link); Pater et al., 2021 (link)). The FastQ files generated by the Mk1C device were used for analysis following the ARTIC Network analysis workflow and EP2ME-lab.

All amplifications were performed on a GeneAmp PCR System 9700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Using the manufacturer’s suggested reaction mix constituents, the Q5 Hot Start High-Fidelity DNA polymerase (New England BioLabs Inc [M0494S], Ipswich, MA, USA) was used for insect amplifications, and the KAPA3G Plant DNA polymerase (KAPA Biosystems [KK7251], Wilmington, MA, USA) for plant and macro-fungi amplifications. The barcode regions targeted for amplification were those adopted and advocated for use by the barcoding community and CBOL: 1) insects, COI [2 ]; 2) macro-fungi, ITS (subunits 1 and 2) [4 (link)]; and 3) plants, rbcL and matK [3 (link)]. Considering alternate loci have been identified by CBOL and are often used as supplemental markers for the identification of land plants, data for the intergenic spacer trnH-psbA and ITS2 were also collected for plant taxa. S1 Table outlines the primer pairs and cycling conditions used in amplifications. Given that the taxa included in this study spanned numerous orders, successful amplification of regions such as matK and trnH-psbA was not possible for all taxa using a single primer pair; amplification of these regions were only achieved after screening with multiple primer pairs. PCR products were screened, purified and quantified as outlined in Meiklejohn et al. (2018) [21 (link)].

Community DNA samples showing PCR amplification with 1.5 Kb band were used for standard PCR for assessing the presence of targeted rumen bacteria using specific bacterial 16S rRNA primers, Q5^® Hot Start High-Fidelity DNA Polymerase (M0493, New England Biolabs), 0.2 mM of each dNTPs (N0446S, New England Biolabs), 100 ng template, and 10 nM of forward and reverse primer (Table-1) [10 ,11 (link)]. PCR was performed in 20 µl reaction volume using the following program: 98°C for 10 min; 35 cycles consisting of 98°C for 10 s; optimized annealing temperature as given in (Table-1) for 30 s; 72°C for 1 min; and a final extension step consisting of 72°C for 10 min. The amplification was determined by electrophoresis of reaction product in 2% agarose gel.
Methods which performed consistently to give intact, good quality DNA with clean PCR bands for all targeted bacteria were finalized further for 16S rRNA gene sequencing and identification studies.