Biotaq dna polymerase

For each sample, each microsatellite locus was amplified by individual polymerase chain reaction (PCR). All amplification reactions were performed in 25 µl volume containing 10 ng of template genomic DNA, 1x NH₄ reaction buffer (16 mM (NH₄)₂SO₄, 67 mM Tris-HCl pH 8.8, 0.01% stabilizer; supplied with BIOTAQ DNA Polymerase), 3 mM MgCl₂, 1 µM of each primer (primer type depended on the type of sequencing technique; for details see above), 200 µM of each dNTP and 1 Unit BIOTAQ DNA Polymerase (Bioline). PCR conditions were 94°C for 5 min followed by 32 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min. The PCR products were verified on a 1% agarose gel stained with SYBR Safe.
All successfully amplified PCR products were purified using Agencourt AMPure XP DNA purification kit following the manufacturer's instructions (Beckman Coulter). DNA concentrations of the purified amplicons were measured using Quant-iT PicoGreen dsDNA Assay kit (Invitrogen, Molecular Probes).

For the identification of Leishmania species, we amplified the ribosomal ITS-1 region with primers LITSR (5'-CTG GAT CAT TTT CCG ATG-3') and L5.8S (5'-TGA TAC CAC TTA TCG CAC TT-3') [46 (link)]. Amplification reactions were performed in volumes of 50 μl containing 3 μl of isolated DNA, 5 μl of 10× buffer (BIOTAQ DNA Polymerase, Bioline, London, UK), 1.5 mM MgCl₂, 0.2 mM dNTP, 0.2 mM of each primer and 1.5 units of Taq polymerase (BIOTAQ DNA Polymerase, Bioline). A denaturing step at 95 °C for 2 min, followed by 35 cycles of denaturing for 20 s at 95 °C, annealing for 30 s at 53 °C, and extension for 1 min at 72 °C, followed by a final extension at 72 °C for 1 h was carried out in thermal cycler (MJ Research PTC-200 DNA Engine, Alameda, CA, USA). DNA samples extracted from promastigote cell cultures of L. infantum, L. tropica, L. major and L. braziliensis were used as positive controls. A non-template control with the same reagents described above but without DNA was added to PCR to rule out contamination.
The PCR products, previously digested with the restriction enzyme BsuRI (HaeIII), were separated by electrophoresis in 2% wide-range agarose (Sigma) at 150 V in SGTB 1× buffer (GRISP LDA, Research Solutions, Porto, Portugal). A solution of SYBR safe DNA gel stain (Invitrogen Ltd., Paisley, UK) was used to visualize the separated DNA fragments under UV light [47 (link)].

Knockout (KO) mutants for biofilm-associated genes were constructed according to a previously described method [85 (link)] with some modifications. Briefly, the genes and their flanking regions were amplified by PCR using the proofreading enzyme ACCUZYME^TM DNA polymerase (Bioline, Memphis, TN, USA), 5′-A tailed using BIOTAQ^TM DNA polymerase (Bioline, Memphis, TN, USA) and cloned in the commercial cloning vector pGEM-T Easy (Promega, Madison, WI, USA). The resulting plasmids were linearized either by an outward PCR performed with BamHI cutting site containing primers (pGflaAB) or by restriction enzyme digestion with MunI, ClaI, BmtI or AflII (pGfliS, pGluxS, pGpta and pGspoT, respectively). The linearized plasmids were ligated to a kanamycin (Km) resistance cassette (aph(3′)-III) obtained from the pMW2 plasmid [86 (link)] either by BamHI digestion or by PCR amplification using primers that contained the appropriate restriction cutting site for each case. The orientation of the cassette and the ORFs was the same in all the constructed plasmids.