This was also a central theme in the DttP
issue on the importance of the tangible legacy collection.
With 4 million operational square feet available, the DTTP
will support the design, engineering and assembly of a wide array of energy-efficient heating and cooling products for residential and commercial use, marketed under the Daikin, Goodman and Amana brands.
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].
PCR was performed in a total volume of 20 L using 2.5 L (15 ng L-1) of genomic DNA; 10X PCR buffer without MgCl2 (10 mM Tris-HCl, 50 mM KCl, pH 8.3); 3 mM MgCl2; 0.1 mM each of dATP, dGTP, dCTP, and dTTP
; 0.5 units of Taq DNA polymerase; and 0.15 mM of each primer.
Total 50 ml reaction volume was made for PCR containing 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 3 mm MgCl2, 100 mm, each of dATP, dCTP, dGTP, dTTP
, 30 mg of primer, 0.001% (w/v) gelatin, 20 ng of genomic DNA and 2 units of Taq polymerase.
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.
Incubation with 20 and 40 [micro]M EGCG resulted in a depletion of intracellular dATP pools to 51 and 37% of controls, respectively, whereas dTTP
pools were increased to 141% of untreated cells upon 40 [micro]M EGCG (Fig.
Unless otherwise indicated, we performed PCR in 5-[micro]L reaction volumes containing 50 mmol/L Tris (pH, 8.3), 3 mmol/L Mg[Cl.sub.2], 200 [micro]mol/L of each dNTP (dATP, dCTP, dGTP, dTTP
), 500 [micro]g/mL nonacetylated BSA (Sigma), 2% (vol/vol) glycerol (Sigma), 50 ng purified human genomic DNA, and 1 x LCGreen[R] Plus (BioFire Diagnostics).
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).
Amplification reactions were carried out in triplicate in a final volume of 20 that contained 3 of bisulfite-modified DNA; 600 nM concentrations of forward and reverse primers; 200 nM probe; 0.6 U of platinum Taq polymerase (Invitrogen, Frederick, MD); 200 mM concentrations each of dATP, dCTP, dGTP, and dTTP
; and 6.7 mM Mg[Cl.sub.2].
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