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(13,14) For this purpose, a 5-[micro]L bacterial DNA template, 2.5 [micro]L Taq buffer, 1.5 mM Mg[Cl.sub.2], 200 [micro]M dNTP, 20 pmol forward and reverse primers, 1.25 U Taq polymerase were prepared in 25 [micro]L volume.
The PCR to amplify the target genes on the cDNA was carried out under the following conditions: 1.8 [micro]L dNTP, 2 [micro]L 10x buffer, 0.2 [micro]L Taq, and 12 [micro]L distilled water were added to 2 [micro]L, 50 ng/[micro]L diluted DNA and 5 [micro]mol/[micro]L diluted forward primer and reverse primer.
The second step was performed in a 50-[micro]L reaction mixture containing 3 [micro]L of the PCR product obtained in the first reaction, 0.48 [micro]M of each second step primer, 0.2 [micro]M each dNTP, 1.50 mM MgCl, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, and 0.5 U Taq DNA polymerase.
PCR was performed with 15 nL reactions containing 50 ng genomic DNA, 10 [micro]M of each primer (foiward and reverse), 400 [micro]M of each dNTP (dATP, dGTP, dCTP, and dTTP), 0.50 units of Taq polymerase in buffer (Promega), and 3 mM of Mg[Cl.sub.2].
PCR reactions were performed in a total volume of 25 [micro]L, including 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 200 [micro]M dNTPs each (Fermentas, Germany), 25 pmoL of each primer, 1.5 U of Taq DNA polymerase (Fermentas, Germany), and 3 [micro]L (40-260 ng/[micro]L) of DNA.
Reactivos Concentracion final 1X ([micron]L) Agua HPLC 6.3 Buffer PCR 5X 1X 3.0 dNTP's 10mM 200 [micron]M 0.3 Oligo Forward 10mM 200 [micron]M 0.3 Oligo reverse 10mM 200 [micron]M 0.3 Taqpolimerasa 5U/[micron]L 2 U 0.24 Muestra ADN 10-50 ng/[micron]L 3.0 Volumen final 15 [micron]L Cuadro 3.
Taking the extracted total DNA as a template, PCR of 16S rDNA gene was made by using the primer mix 520F: (5-CCTACGGGNGGCWGCAG-3) and 802R: (5-GACTACHVGGGTATCTAATCC-3) having specificity to V3-V4 regions of 16S rDNA gene to make PCR amplification; the amplification reaction system (50 uL) include: 50 ng genomic DNA template and 2 uL dNTP (2.5 mmol/L, TakaRa); the concentration of both primers were 0.4 mol/L; 0.5 uL DNA Taq polymerase (1.5U, TaKaRa); 5 uL 10x PCR Buffer.
PCR was performed in 25 [micro]l reaction volumes containing 0.4 [micro]M of each primer, 250 [micro]M of each dNTP, 1U Taq DNA polymerase with 1.5 mM Mg[Cl.sub.2], and 50 ng genomic DNA.
Instruments include traditional and real-time thermocyclers while reagents and consumables include enzymes, dNTP's, template DNA, primers and probes, buffers, master mixes, nuclease free water and others (tracking dye and wax beads).
For the first round PCR, 5 [micro]l of purified DNA was added to the PCR mixture (final volume of 50 [micro]l) containing 3 [micro]l of both outer primers (25 pmol/L), 10 [micro]l 5xPCR buffer (including 1.5 mmol/L MgCl [sub]2), 1 [micro]l l0 mmol/L dNTPs (2.5 mmol/L of each dNTP), 1.5 U PromegaGoTaq polymerase, and water to 50 [micro]l.
The PCR amplifications were performed using about 1[micro]g of fungal template DNA, 200 pmol of each primer (Nor1-Forward: 5 -ACC GCT ACG CCG GCA CTC TCG GCA C-3' and Nor2-Reverse: 5 -GTT GGC CGC CAG CTT CGA CAC TCC G-3'), Mg[Cl.sub.2] -free reaction buffer, 2.0mM Mg[Cl.sub.2], 2.5U of Taq polymerase and 0.2mM of each dNTP. PCR was carried out under the following conditions: one cycle at 94[degrees]C for 3min; 30 cycles at 94[degrees]C for 1min, at 55[degrees]C for 1min and at 72[degrees]C for 1min; and at 72[degrees]C for 10min in a final extension.
The DNTP within the NIEHS is conducting research along with various agencies, including the US Environmental Protection Agency, the US Army, the National Human Genome Institute, and the Centers for Disease Control in the National Toxicology Project.
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