Deoxynucleotide triphosphates, solution

The first step of a PCR involves DNA denaturation at 90-95 °C, in a buffered, neutral, aqueous solution containing DNA polymerase, the four deoxynucleotide triphosphates and Mg++, in the presence of a large excess of the two primers (Fig. 27). In the second step, the temperature of the reaction is lowered to about 10 °C below the melting temperature of the primers and the primers (which are considerably smaller than the DNA) are allowed to hybridize to their complementary sequence on the DNA template molecule. This temperature is still too high for the DNA to fully renature. The temperature is then raised to 72 °C, the optimal temperature for extension of the primers by the DNA polymerase, which catalyses the addition of nucleotide triphosphates to extend the sequence in each direction from the... 

Hi) Deoxynucleotide triphosphate (dNTP°) mixes Stocks of 20 mM dNTP s (in 5mM Tris-HCl, pH 7.4, 0.1 mM EDTA) are stored frozen. The 0.5 mM working solutions are made freshly by dilution into water. 

Add 1 /xl CTAG mix (equal vols. of 20 mM solutions of the four deoxynucleotide triphosphates). 

B2518), and 5 units/mL Klenow fragment of DNA polymerase I. The labeling solution without polymerase can be prepared in bulk and stored frozen in aliquots, but the DNA polymerase must be added immediately prior to use. The biotinylated nucleotide can be obtained from Life Technologies Ltd. (Paisley, Scotland cat. no. 19534-016). Deoxynucleotide triphosphates can be purchased as a set from Boehringer Mannheim UK, East Sussex, UK cat. no. 1277 049 (see Notes 4,5, and 7). 

PCR is performed in a total volume of 25 juL. The reaction mixture contains 0.2 mM of each deoxynucleotide triphosphate, 0.2 pM of each primer, 1 x PCR buffer (GeneAmp PCR buffer. Applied Biosystems), 2 mM MgCl2,1.5 units Taq polymerase (AmpliTaq Gold DNA Polymerase, Applied Biosystems), and 2 pL (30 to 100 ng) of template DNA solution. Primers and PCR conditions are listed in Tables 7.1 to 7.3. 

Deoxynucleotide triphosphates (dXTPs) Make up a single solution containing 2.5 mM of dATP, dGTP, dCTP, and d lTP. 

Deoxynucleotide triphosphates or dNTPs These can be obtained from any reliable source. We routinely use neutralized, 100 mM stock solutions from Pharmacia, Piscataway, NJ. Store frozen at -20 C. 

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