Neb q5 high fidelity dna polymerase

Genomic DNA of strain ESM356 (wild-type) was isolated using a lithium acetate-SDS based protocol as previously described (Looke et al., 2011 (link)). PCR amplification of the desired fragments was performed using NEB Q5 High-Fidelity DNA polymerase (#M0491S, NEB). The PCR fragments were assembled into the linearized vector pRS305 (integration vector; Sikorski and Hieter, 1989 (link)) using NEBuilder HiFi DNA Assembly reaction mix (#E2621L, NEB) as per the manufacturer's instructions. The reaction product was transformed into Escherichia coli TOP10 cells (#C404010, Invitrogen) and plasmid isolation was performed using the Thermo Fisher GeneJet Miniprep Kit (#K0503, ThermoScientific). The positive transformants were confirmed using restriction digestion and sequencing. ESM356 (S288c background) was used as a wild-type strain and all subsequent strains were derived from it. Yeast culturing and transformation were undertaken using established protocols. Yeast expression plasmids carrying mNG–Tpm1 and –Tpm2 (piSP347 and piSP349, respectively) were linearized with the KasI restriction enzyme and transformed for integration into the leu2 locus in the yeast genome as a second copy. The positive transformants were selected on SC-Leu plates and expression of mNG–Tpm1 and –Tpm2 were confirmed using fluorescence microscopy.

Loci coding sequences were extracted from the contig files using BIGSdb incorporating flanking regions. Consensus sequences were generated to facilitate primer design. Sequences were loaded into NCBI Primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) with settings to ensure the whole coding sequence would be amplified. All primer pairs where chosen for compatible annealing temperatures to allow for all reactions to use the same amplification conditions (Table 3). All PCR amplifications were conducted in a 50 μl reaction volume using 50 ng of purified D. nodosus chromosomal DNA as template. All reactions were assembled on ice using a final concentration of 200 μM dNTPs, 0.5 μM of forward primer and 0.5 μM or reverse primer (Table 3), 0.02 U/μl of NEB Q5 High-Fidelity DNA Polymerase (New England Biolabs Inc., U.S.A.) in 1x Q5 reaction buffer. A preheated Thermocycler was used with an initial denature of 98°C for 30 s followed by 35 cycles of 98°C for 10 s, 60°C for 25 s, and 72°C for 30 s. A final extension of 2 min at 72°C was used before a hold of 10°C.

RNA isolation of P. thermosuccinogenes transformants, harboring the pThermoCas9i plasmids was performed using 5 mL of overnight cultures at an OD₆₀₀ ~ 1.0. The RNA isolation for qRT-PCR was adapted from Ganguly et al. [28 (link)]. The Maxwell 16 LEV Total RNA Cells Kit was used to obtain RNA from the transformants. The quality and the concentration of the purified RNA were determined using a NanoDrop spectrophotometer and the material was stored at −20 °C. The first strand cDNA synthesis was performed with SuperScript^TM III Reverse Transcriptase (Invitrogen) following the manufacturer’s instruction. The primers BG11642 and BG11643 were used to amplify 169 bp of the sgRNA and the primers BG11636 and BG11637 were used to amplify 282 bp of the ThermodCas9 using the NEB Q5^® High-Fidelity DNA polymerase.

Genomic DNA was extracted from 3 × 10⁶ HEK293_LP^CD19/BiTE cells using Quick-DNA midiprep kit (#D4075, Zymo Research). Gene-specific primers annealing to the signal sequence (5’-CTGGTCCTGCATCATCCTGTTTC-3’) and P2A sequence (5’-GTTAAAGCAAGCAGGAGACGTGG-3’) were used to amplify BiTE variants from the genomic DNA. The amplification was carried out with 500 ng of template genomic DNA (corresponding to a gene copy number of 145,000) with an annealing temperature of 69 °C and extension time of 1 min 30 s, using NEB Q5 High-Fidelity DNA Polymerase (#MO491, New England Biolabs Inc.). The genomic region containing integrated BiTE sequences amplified by PCR was gel purified and analyzed by long-read amplicon sequencing (PacBio, SMRT). The circular consensus reads (~3 million CCS reads) from long-read amplicon sequencing was parsed for gene sequences containing both N-terminal signal sequence and C-terminal his-tag sandwiching the BiTE region using bioinformatics services provided by vendor FornaxBio (Worcester, MA). These gene sequences were translated and analyzed for unique and duplicated sequences. The unique BiTE sequences were additionally sorted by linker type, VH–VL orientation of CD19 and CD3 scFvs, and representation of scFv candidates. The sequence analysis was carried out using scripts generated in R using standard library packages (stringr, Biostrings).

Partial panC gene was amplified using following primer sequences for groups I to VI according to Guinebretière et al. (2010) (link): 5′-TYGGTTTTGTYCCAACRATGG-3′ (forward degenerated primer) and 5′-CATAATCTACAGTGCCTTTCG-3′ (reverse primer). PCR was carried out in a Biometra Trio 48 thermocycler (Analytik Jena, Germany) in a final volume of 25 μl containing 1X NEB Q5 Reaction Buffer (New England Biolabs, Ltd, Ipswich, United States), 200 μM of each dNTP, 0.5 μM of each primer, 0.5 IU of NEB Q5 High Fidelity DNA Polymerase (New England Biolabs) and 1 μl DNA template. The temperature protocol included an initial denaturation at 98°C for 30 s followed by 30 cycles of 98°C for 10 s, 61°C for 30 s, 72°C for 30 s and a final extension at 72°C for 2 min. The INVISORB Spin DNA Extraction Kit (Invitek, Berlin, Germany) was used for DNA purification and products were sent to Eurofins Genomics Germany GmbH (Ebersberg, Germany) for sequencing using primer 5′-ATAATCTACAGTGCCTTTCG-3′ (Guinebretière et al., 2010 (link)). Sequence data analysis was done by uploading to an online algorithm¹ for assignment to groups I to VII (Guinebretière et al., 2010 (link)).