The Department of Tourism and Commerce Marketing (DTCM) is to enable the hospitality industry in Dubai to become more sustainable and efficient in managing their resources with the launch of Dubai Green Tourism Programme (
DGTP).
Para a reacao da polimerase em cadeia, foi utilizado o Pre-mix IC (2x) da Phoneutria contendo tampao IC (50 mM KCl, 10mM TrisHCl pH 8,4 e 0,1 TritonX-100, Mg[Cl.sub.2] 2,0 mM), nucleotideos (dATP, dCTP,
dGTP, dTTP) e enzima termoestavel Taq DNA Polimerase.
The final volume was 50[micro]L, containing 5[micro]L DNA, 1X PCR Buffer (500mM KCl, 100mM Tris-HCl [pH 9.0]); 1[micro]L (200[micro]M each) dATP, dCTP,
dGTP e dTTP; 20pmol from each primer; 2.5U Taq DNA polymerase and 2mM Mg[Cl.sub.2].
Reaction mixtures contained 0.4 [micro]M (each) primer, 0.2 pM (each) deoxynucleoside triphosphate (dATP,
dGTP, dTTP and dCTP) from CinnaGen-Iran, and 1X PCR reaction buffer 1.5 [micro]M, Mgcl2 with the addition of 10 [micro]l of the extracted DNA.
The primary amplification was performed by using the oligonucleotide primers PfATPase6P1 and PfATPase6-P2 as described elsewhere (4) in a reaction mixture of total volume 20 [micro]l which consisted of 5 pl of genomic DNA, 4 [micro]l of 5 x colourless Gotaq reaction buffer, 0.3 [micro]M (0.6 [micro]l in 20 [micro]l of reaction volume) of each primer, 0.2 mM (0.165 [micro]l in 20 [micro]l of reaction volume) of each deoxyribonucleoside triphosphate (dATP, dTTP,
dGTP, dCTP) and 1.2 units of taq DNA polymerase (0.24 [micro]l in 20 [micro]l of reaction volume).
The amplification of the 16S rRNA genes was performed in a 50 mL reaction containing 1 mL nucleic acid extract, 1 unit of Platinum Taq High Fidelity polymerase (Invitrogen, Carlsbad, CA, USA) or Immolase Taq (Bioline, Randolph, MA, USA), the manufacturer's PCR buffer, 2.0 mmol/L MgS[O.sub.4], 200 [micro]mol/L dATP, 200 [micro]mol/L dCTP, 200 [micro]mol/L dTTP, 200 [micro]mol/L
dGTP (Bioline), and 250 nmol/L of each primer.
The PCR reaction was settled down by adding template DNA 40ng and dNTP 100M with (0.2 Mm of each
dGTP, dCTP, dTTP and dTTP),1.5 mM MgCl2, individual primers (forward and reverse)1.0 M, 1X PCR buffer (50 mM KCl, pH 8.310 mM Tris- HCl) and 0.2 U Taq polymerase.
All reaction mixtures contained 20 nM of the template, 10 nM of each primer, and 100 [micro]M of each of the four dNTPs (dATP, dCTP,
dGTP, and dTTP).
When the following 2 bases, dTTP and dATP, are dispensed, they cannot be used to extend the nascent DNA strand, whereas peaks are seen when
dGTP and dTTP are dispensed.
DNA amplification reaction was performed in a volume of 20u l containing DNA 1u l (10ng/u l), primer 4u l (5pmol/u l), PCR buffer 2u l, MgCl2 1.35u l (1.6mM), Taq DNA polymerase (Fermentas) 0.3u l (5u /u l), dNTPS 10u l (0.5m M each of dATP, dTTP, dCTP and
dGTP), H2O 0.35u l.
The PCR reactions included approximately 100 ng of genomic DNA, 2.5 [micro]l of 10x buffer (Tris-HCl, pH 9.0), PCR enhancers, (N[H.sub.4])2S[O.sub.4], 20 mM Mg[Cl.sub.2]), 2.0 [micro]l of 10 mM dNTPs mixture (2.5 mM each of dATP, dCTP,
dGTP and dTTP), 1 of 10 pmol of each primer and 1 U HS Prime Taq (GeNet Bio, Korea) in a 25 [micro]l reaction volume.