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.
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.