Pfu ultra dna polymerase

To generate the cysB⁺ strain TIM409, the gene VF_1490 was amplified with its promoter region by PCR from ES114 genomic DNA using the primers indicated in Table 2. The resulting amplicon was cloned into pCR-Blunt (Life Technologies, Carlsbad, CA, USA) and then sub-cloned using KpnI/SpeI restriction sites into pEVS107 to yield pTM420. The cysB⁺ allele was integrated into the large chromosome of the ΔcysB strain NPW003 at the Tn7 site as described elsewhere (Miyashiro et al., 2011 (link)). To generate the cysBA227D allele, the pCR-Blunt clone containing the cysB⁺ allele was used as a template for PCR-based, site-directed mutagenesis with the primer set listed in Table 2. The reaction contained PFU Ultra DNA Polymerase (Agilent Technologies, Santa Clara, CA) with 0.2 mM dNTPs, 125 ng each primer, and 20 nM plasmid template. Amplification was performed in a C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) with the following steps: 1) 95°C/30 s, 2) 95°C/30 s, 3) 55°C/1 m, 4) 68°C/ 10 m, and 5) 18 cycles of steps 2–4. The PCR product was digested with 10 U DpnI overnight and transformed into Top10 by electroporation. The resulting cysBA227D was sub-cloned into pEVS107 to yield pTM423 and then integrated into the Tn7 site of NPW003 as described above.

DNA manipulations were performed as in (Sambrook et al., 1989 ). E. coli S17-1λpir was used for cloning. iProof DNA polymerase (Bio-Rad) was used for regular PCR reactions, and PfuUltra DNA polymerase (Agilent) was used for constructing point mutations. Restriction enzymes, T4 polynucleotide kinase, Antarctic phosphatase, and T4 DNA ligase were purchased from New England Biolabs (NEB). Plasmids used in this study are described in Table S2. Primers from Integrated DNA Technologies (IDT) are listed in Table S3. All plasmids were confirmed by sequencing at Genewiz. Arabinose inducible qrr4, anhydrotetracycline inducible qrr4, and target-GFP translational fusions were constructed as described (Shao et al., 2013 (link)). V. cholerae mutants were constructed as described (Skorupski and Taylor, 1996 (link)).

Plasmids pNL43/XmaI, pUC19.2LTR, pNLX.Luc.R- [31 (link)], pCG-VSV-G, pCG-gagpol, pIRES2-eGFP, pIRES2-eGFP-LEDGF-HA [10 (link)], pKBIN6Hthr [48 (link)], and pFT-1-LEDGF [19 (link)] were previously described. Plasmid pLPCX-HA-LEDGF/p75 was built by inserting PCR-amplified HA-LEDGF/p75 into pLPCX vector DNA (Clontech Laboratories, Inc., Mountain View, California). Mutations were introduced by PCR using Pfu Ultra DNA polymerase (Agilent Technologies, Inc., Santa Clara, California), and plasmids were sequenced to verify the presence of mutations and absence of unwanted secondary changes.