Taq dna polymerase

To amplify DNA fragments for cloning and screening, we used Phusion Hot Start DNA polymerase II (Thermo Scientific) and Taq DNA polymerase (Denville Scientific), respectively. We used T4 DNA ligase (Thermo Scientific) for blunt-end ligations. If applicable, we treated purified PCR products with DpnI (Thermo Scientific) to remove the plasmid template DNA. Phosphorylation of double-stranded DNA (dsDNA) fragments was performed with T4 polynucleotide kinase (Thermo Scientific). Ligase cycling reactions (LCRs) were performed as described previously (43 (link)).

All restrictions enzymes were purchased from New England Biolabs (NEB, USA), whereas the lysozyme from chicken egg white and mutanolysin from Streptomyces globisporus were purchased from Sigma-Aldrich (Sigma-Aldrich, USA). Phusion polymerase (NEB, USA) was used to generate PCR amplicons for Sanger sequencing, whereas Taq DNA polymerase (Denville Scientific, USA) was used for screening purposes. The oligonucleotides used in this study are listed on Additional file 5: Table S2 (Integrated DNA Technology-IDT, USA).

Plasmid DNA was isolated from 4-ml B. burgdorferi cultures (1×10⁷ to 1×10⁸ cells/ml) with the Qiaprep Spin Miniprep kit (Qiagen) and resuspended in 30 µl of nuclease-free water. PCR primers (Table 4) were designed to amplify a 2.5-kB sequence on lp36, encompassing bbk42–bbk45[7] (link), [62] (link). Each PCR mixture contained approximately 10 ng DNA, 250 µM deoxynucleoside triphosphates (Roche), 10 ng of each primer and 1 U Taq DNA polymerase (Denville Scientific, Inc.). PCR conditions were as follows: initial denaturation at 95°C for 3 minutes; 35 cycles of 95°C for 1 minute, 60°C for 40 seconds, 72°C for 3 minutes; and final extension at 72°C for 2 minutes. PCR of bbk19 was performed to assess the presence or absence of lp36. PCR parameters were as follows: initial denaturation at 95°C for 3 minutes, followed by 35 cycles of 95°C for 30 seconds, 48°C for 30 seconds, 72°C for 3 minutes, and final extension at 72°C for 2 minutes. ospA (bba15) was amplified as a control for the presence of plasmid DNA using the following conditions: initial denaturation at 95°C for 3 minutes; followed by 35 cycles of 95°C for 30 seconds, 52°C for 30 seconds, 72°C for 3 minutes; and final extension at 72°C for 2 minutes. PCR products were analyzed by electrophoresis in 1% agarose gels prepared in 1X TBE buffer and stained with ethidium bromide.