Journal: AoB Plants

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline

doi: 10.1093/aobpla/pls033

Figure Lengend Snippet: Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp DNA size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.

Article Snippet: All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume.

Techniques: Amplification, Marker, Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: AoB Plants

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline

doi: 10.1093/aobpla/pls033

Figure Lengend Snippet: Alexandrium tamarense microsatellite amplifications of the three local strains with (A) specific primers ATB8 (lanes 1, 2 and 3) and ATD8 (lanes 4, 5 and 6). Lanes 7 and 8 correspond to controls with primers ATB8 without DNA. (B) Specific amplification with primers ATB1 (lanes 1, 2 and 3) and ATF11 (lanes 6, 7 and 8). Lanes 4–5 and 9–10 are controls without DNA for primer sets ATB1 and ATF11, respectively. M = 100-bp DNA size marker. Species-specific microsatellite amplifications using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller TM 100-bp DNA ladder was used for size estimation of amplified fragments.

Article Snippet: All amplifications were carried out in duplicate with 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of recombinant Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) in a 10-µL reaction volume.

Techniques: Amplification, Marker, Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: The EMBO Journal

Article Title: DNA polymerase mu (Pol ?), homologous to TdT, could act as a DNA mutator in eukaryotic cells

doi: 10.1093/emboj/19.7.1731

Figure Lengend Snippet: Fig. 6. Pol μ-catalysed misinsertion at the four template bases. The four template–primer structures used, which differ only in the first template base (outlined), are indicated on the left. The single-stranded oligonucleotide corresponding to the primer strand was assayed in parallel as a control of DNA-independent nucleotide insertion. Mg 2+ -activated nucleotide insertion on each 5′–labelled DNA substrate (3.2 nM) was analysed in the presence of either the complementary nucleotide (10 μM) or each of the three incorrect dNTPs (100 μM), as described in Materials and methods. Mn 2+ -activated nucleotide insertion was assayed with each of the four dNTPs (0.1 μM). After incubation for 15 min at 30°C in the presence of 20 ng of human Pol μ, extension of the 5′–labelled (*) strand was analysed by electrophoresis in an 8 M urea–20% polyacrylamide gel and autoradiography.

Article Snippet: The PCRs were performed in a total volume of 10 μl, using 25 ng of template, 5 μM primers h2MAPs and h2MAPas, 1.25 mM MgCl2 , 0.2 mM dNTPs and 0.025 U of Taq DNA polymerase (Gibco-BRL) in the buffer supplied by the manufacturer.

Techniques: Incubation, Electrophoresis, Autoradiography

Journal: The EMBO Journal

Article Title: DNA polymerase mu (Pol ?), homologous to TdT, could act as a DNA mutator in eukaryotic cells

doi: 10.1093/emboj/19.7.1731

Figure Lengend Snippet: Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and dTTP, in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.

Article Snippet: The PCRs were performed in a total volume of 10 μl, using 25 ng of template, 5 μM primers h2MAPs and h2MAPas, 1.25 mM MgCl2 , 0.2 mM dNTPs and 0.025 U of Taq DNA polymerase (Gibco-BRL) in the buffer supplied by the manufacturer.

Techniques: Inhibition, Incubation, Concentration Assay