Al Rifai added that DGTP
would detail 'no cost' and 'low cost' options for hoteliers seeking to apply sustainability solutions for 'quick wins'.
The Invitrogen kit (Life Technologies/USA) was used for the reaction, with 1X PCR Buffer (500mM KCl, 100mM Tris-HCl [pH 9.0]); 5[micro]L (1mM each) dATP, dCTP, dGTP
e dTTP; 0.5[micro]L from each primer, 1.0U Taq DNA polymerase and 2mM Mg[Cl.sub.2].
However, each of these reaction mixtures contained 20 nM of the template, 10 nM of each primer 5' -TATTGATTGTGAATTA(C/G)-3', and 100 [micro]M of a single dNTP (dCTP, dGTP
, dATP, or dTTP).
For codons 12, 13, and 14 of wild-type KRAS and 12b KRAS mutant (G [right arrow] A), both molecules are elongated when dGTP
is dispensed, but the nascent strands are out of phase because the strand replicating the wild-type allele incorporates 2 dGTPs
, whereas the one replicating the mutant allele only incorporates one dGTP
(Figures 5, A, and 7, A).
As previously described  PCR was performed in a final reaction volume of 100 [micro]L with 5 [micro]L of sample material and 95 [micro]L of kit working master mix (containing Mg[Cl.sub.2], KCl, AmpliTaq, gold DNA polymerase (AmpliTaq; Perkin-Elmer; Foster City, CA), uracil-N-glycosylase, dATP, dCTP, dGTP
, dUTP, dTTP, and biotinylated PGMY primers and [beta]-globin primers GH2O and PCO4).
The PCR reaction mixture consisted of 1200 nM of each primer, 200 nM probe, 0.4 U AmpliTaq gold polymerase, 200 nM each of dATP, dCTP, dGTP
, and dTTP, 3.5 mM Mg[Cl.sub.2], and 1 x Taqman buffer A containing a reference dye.
100ng of DNA was dissolved in a 25[micro]l system that included 1.1 [micro]mol/LMg[Cl.sub.2], 200 [micro]mol/ LdNTP(dATP, dCTP, dGTP
, dTTP), 1[micro]mol/L primers, and 1.5uTaq enzyme.
Polymerase chain reactions (PCRs) were performed for each individual in a 50-[micro]l total volume consisting of the following: 5[micro]l of DNA (10 ng/ml) from the individual; 1.5 [micro]l of a 10-mM solution of each primer (P-2 and P-8; Griffiths et al., 2002); 5[micro]l of a dNTP mixture containing 1 mM each of dATP, dCTP, dGTP
, and dTTP; 2[micro]l of a 25-mM solution of magnesium chloride; 0.2 [micro]l of Taq DNA polymerase (Promega: GoTaq Flexi DNA Polymerase; Promega Corp., Madison, Wisconsin); 10 [micro]l of a 5X reaction buffer (Promega); 24.8 [micro]l of sterile water.
A 2 [micro]L volume of DNA template was added to 23 [micro]L of reaction mixture, which contained 2.5 [micro]L of PCR buffer 10x (10 mM Tris HCl, 25 mM KCl), 1mM of Mg[Cl.sub.2], 0.5 [micro]M of each primer, 100 [micro]M of each deoxynucleotide trifosfate (dATP, dGTP
, dCTP, dTTP) and 1.5U of Taq polimerase (Invitrogen--Sao Paulo, Brazil).
Labeling was obtained using a PCR procedure with the following dNTP concentrations: 200 [micro]M dATP, 200 [micor]M dCTP, 200 [micro]M dGTP
, 150 [micro]M dTTP, and 50 [micro]M dig-11-dUTP (Roche Molecular Biochemicals).
Condicoes de amplificacao do DNA: Preparou-se uma mistura de reagentes em um microtubo para capacidade para 200 [micro]L que consistiu em 1, 5x de tampao Taq, 4 mM de Mg[Cl.sub.2], 0, 2 [micro]M de cada oligonucleotideo (G1 e G2), 100 [micro]M de dNTP (dATP, dTTP, dCTP e dGTP
) e 2,5U, de Taq DNA Polimerase (Ludiwing [R]).
Polymerase Chain Reactions (PCR) were carried out in a final volume of 25 [micro]l containing 20 ng template DNA, 100[micro]M each dATP, dTTP, dGTP
& dCTP, 20 ng of decanucleotide primers, 1.5mM Mg[Cl.sub.2], 1 x taq buffer (Fermantas) [10mM Tris-HCl (pH 9.0), 50mM KCl, 0.01% gelatin] and 0.5 U Taq DNA polymerase (Fermentas).