Velocity dna polymerase

DNA manipulations were performed as described in Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Enzymes for molecular biology were obtained from Fermentas and Thermo Fisher. PCRs were performed with using Velocity DNA polymerase (Bioline). Oligonucleotides employed for strain and plasmid construction are listed in Supplementary Table 7. Information regarding construction strategies is available upon request. Plasmids used in this study carry the ampicillin resistance gene for selection in E. coli and are listed in Supplementary Table 7.

Amino acid sequence of BIF protein [1 (link)] was translated according to the standard genetic code into the coding nucleotide sequence with codon optimization for the expression in Escherichia coli The DNA fragment encoding this sequence was synthesized by polymerization reaction with overlapping oligonucleotides using the Velocity DNA polymerase (“Bioline”, Luckenwalde, Germany) and then amplified by the polymerase chain reaction (PCR). The obtained PCR fragment was cloned into NdeI and XhoI restriction sites into the pET23b expression vector (“Novagen”, Madison, WI, USA) under the control of T7 RNA-polymerase promotor. The presence of the insert was confirmed by the restriction and sequence analysis. At the C-terminus this construct contained the (His)₆-tag for protein purification by affinity chromatography. Previously, the recombinant analogues of native BJP were studied in a number of works and they reliably reflected the structural peculiarities of native proteins [22 (link),23 (link)].

A range of 13 high fidelity, regular, economy and premium Taq polymerase enzymes were selected: Biotaq® (Bioline, London, UK), FastStart® High Fidelity PCR System (Roche, Mannheim, Germany), AmpliTaq Gold® (Applied Biosystems, Warrington, UK), HotStarTaq® DNA Polymerase (Qiagen, Hilden, Gernamy), Phusion® High Fidelity DNA Polymerase (Finnzymes, Espoo, Finland), Taq DNA Polymerase (Roche, Maylan, France), i-Max^TM II DNA Polymerase (iNtRON Biotechnology, Seongnam, Korea), KAPA HiFi™ (Kapa Biosystems, Boston, USA), OneTaq™ DNA Polymerase (New England Biolabs, Hitchin, UK), Vent® DNA Polymerase (New England Biolabs, Hitchin, UK), Deep Vent® DNA Polymerase (New England Biolabs, Hitchin, UK), Pwo® DNA Polymerase (Roche, Maylan, France) and Velocity DNA Polymerase (Bioline, London, UK) (abbreviated names in Table 1). The list price of these enzymes for the amount recommended for a single 10 μl reaction (not including tax, handling or shipping) ranged from €0.01 to €0.63 (Spain, June 2013).

For genetic complementation of the non-pellicle forming A. baumannii mutants, the genes corresponding to A1S_0112, A1S_0115 and cpdA were PCR amplified, cloned into the shuttle vector pWH1266, transformed into DH5α E. coli cells and sequenced. Plasmids were extracted and subsequently transformed via electroporation using a MicroPulser (Bio-Rad, CA, USA) at 2.5 kV, 200 ohms and 25 microfarads into their respective A. baumannii transposon insertion strains [46 (link)]. PCR was achieved with VELOCITY™ DNA polymerase (Bioline, Vic, Australia) and the oligonucleotides (Additional file 2) using the following PCR protocols. For amplification of cpdA, cycling conditions were 2 min at 94 °C, 30 cycles of 45 sec at 94 °C, 45 sec at 55 °C, and 90 sec at 72 °C, followed by a final extension of 4 min at 72 °C. Amplification of A1S_0112 used the same conditions with the exception of the annealing temperature which was increased to 60 °C. For amplification of A1S_0115, the cycling conditions were 2 min at 94 °C, 30 cycles of 90 sec at 94 °C, 90 sec at 60 °C, 4 min at 72 °C and a final extension of 6 min at 72 °C.

Deletion mutants were generated using the splicing by overlap extension (SOEing) PCR method and allelic replacement, as previously described [18] (link). All primers used in the process are listed in Table 2. Deletion cassettes were cloned into the shuttle vector pKSV7. Double mutants were generated by introducing the deletion cassette into the confirmed single EGDm ΔgadD3 mutant. PCR amplification of DNA for use in cloning and downstream work was carried out by using the high-fidelity Velocity DNA polymerase (Bioline), while screening was carried out by using Biotaq DNA polymerase (Bioline).

The isolated strain which showed to be the most effective for diuron degradation in solution (Figure S1) was identified. DNA was extracted from the liquid cultured of the strain employing the G-spinTM total DNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea), then 16S rRNA gene was amplified by polymerase chain reaction (PCR) using a high-fidelity polymerase (Velocity DNA polymerase from Bioline, Almería, Spain) with universal oligonucleotides primers: 16F27 (annealing at position 8-27 E. coli numbering) and 16R1488 [35 ]. Finally, the PCR product was purified using a NucleoSpin® PCR clean-up gel extraction kit and PCR clean-up (Macherey-Nagel, Cultek S. L., Madrid, Spain) prior to be sequenced.