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Each nested amplification contained 200 [micro]M each dNTP (dATP, dCTP, dGTP, and dTTP), 1.25 units Taq polymerase, and 0.2 [micro]M each of primer.
The DNA was labeled with [[micro]-32p] dCTP by the random-priming method (Pharmacia Biotech).
Before the ASPE reaction, each PCR reaction was treated with shrimp alkaline phosphatase to inactivate any remaining nucleotides (particularly dCTP) so that biotin-dCTP could be efficiently incorporated during the primer extension reaction.
The 25-[micro]L amplification reaction mixture contained 20 mM Tris-HCl, pH 8.3, 50 mM KCl, 3.0 mM Mg[Cl.sub.2], 0.2 [micro]M primer, 0.2 mM each dATP, dGTP, dCTP, and dTTP, 1.5 units Taq DNA Polymerase (GIBCO BRL, Grand Island, NY), and 2 to 5 ng of DNA template.
PCR reactions (25 [micro]L) containing 50 [micro]M each of dATP, dTTP, and dGTP (MBI Fermentas) and 10 [micro]M each of Cy5-dCTP (Amersham Biosciences) and dCTP were subjected to 35 cycles of amplification (94[degrees]C for 30 s, 56[degrees]C for 30 s, and 72[degrees]C for 1 min, with a final elongation of 7 min at 72[degrees]C) as reported previously (2,3).
A reagent mixture containing 50 [micro]M deoxynucleotide triphosphate mixture (dCTP, dTTP, dGTP, and dATP-[alpha]S, treated with pyrophosphatase to remove contaminated pyrophosphate), 1.25 [micro]M SNP genotyping probe (or control), and 0.138 U of Taq DNA polymerase was added to the PCR products.
In addition, the wells contained 1 x PCR buffer (Applied Biosystems); 4 mM Mg[Cl.sub.2]; 250 [micro]M each of dATP, dCTP, dGTP, and dTTP; 0.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems); 2.5 pmol each of both sets of primers; and 1 [micro]L of template DNA in a final volume of 5 [micro]L.
Mr Khaw Boon Wan, co-ordinating minister for infrastructure and minister for transport, and Dr Olumuyiwa Benard Aliu, president of the ICAO council, signed a new memorandum of understanding (M0U) to expand and extend the Singapore-ICAO Developing Countries Training Programme (DCTP) for another three years from 2019 to 2022.
Mr Khaw Boon Wan, Coordinating Minister for Infrastructure and Minister for Transport, and Dr Olumuyiwa Benard Aliu, President of the Council of the ICAO, signed a new Memorandum of Understanding (MOU) to expand and extend the Singapore-ICAO Developing Countries Training Programme (DCTP) for another three years from 2019 to 2022.
From his films distributed on DVDs to his media company DCTP's website that features a rotating constellation of clips from past films and television episodes, Kluge's digital productions are an important component of his oeuvre.
The firm increased production in Phase-II, peaking at 4.5 MCM/d in 2014, when it commissioned the Disouq Central Treatment Plant (DCTP).
Porto, Coimbra: Faculdade de Letras da Universidade do Porto, DCTP, CEAUCP, pp.
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