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The reaction was initiated by activating the CleanAmp dGTP at 95 [degrees]C for 5 min, followed by fluorescence monitoring of nucleotide incorporation at 75 [degrees]C.
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).
5 mM concentrations of dATP, dCTP, dGTP, and dUTP; 20 units of RNasin (Invitrogen), 1 [micro]L T7 RNA polymerase (Invitrogen), 0.
2 mM dATP, dCTP, dGTP and dTTP), 1 U Taq DNA polymerase enzyme (Roche), 10 |iM of forward and reserve primers (Sigma-Genosys), and 10 ng of DNA template.
5, 400 [micro]M dATP, 400 [micro]M dGTP, 400 [micro]M dCTP, 400 [micro]M dTTP, 3 mM MgCl2 [Promega]), 0.
75 mM MgCl2, 350 mM each of dATP, dGTP, dTTP, dCTP, 400 picomoles of primers (wsp-F and wsp-R), 1 unit of Pwo and 5 units of Taq DNA polymerases (Jeyaprakash & Hoy 2000).
The template DNA of unknown sequence is combined in a buffered solution with the four dNTPs (dATP, dCTP, dGTP, dTTP), the four ddNTPs (ddATP, ddCTP, ddGTP, ddTTP), primers and a DNA polymerase (Fig.
1% Triton X-100], 1 unit of Taq polymerase (Banglore Genei), 200 uM (each) deoxynucleoside triphosphate (dATP, dCTP, dGTP, and dTTP) (Banglore Genei), 10 pmol of each primer, 5 [mu]l of sample DNA, and ultra pure sterile water added to 25 [mu]l.
0 [micro]l dNTP [Roche] (10 mM of each deoxyribonucleotide: dATP, dCTP, dGTP, and dTTP), 5.
Using gene-specific primers and template cDNA with a homopolymeric dGTP tail, 5' RACE reactions can be employed to amplify the remaining 5' end of the gene.
All dGTP pools remained beyond the detectability limit of the method.
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.
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