Taq DNA polymerase reaction

Double-stranded products were purified using centrifugation dialysis following the protocol described by Allard et al.31 Purified double-stranded products were subsequently dried under vacuum and resuspended in 15 /A of IX Tris/EDTA buffer (TE). To obtain single-stranded DNA, we used 2 n 1 of the purified double-stranded PCR product as the template. The concentrations of Taq DNA polymerase, reaction buffer, dNTPs, and... 

FIGURE 13.21 Polymerase chain reaction (PCR). Oligonucleotides complementary to a given DNA sequence prime the synthesis of only that sequence. Heat-stable Taq DNA polymerase survives many cycles of heating. Theoretically, the amount of the specific primed sequence is doubled in each cycle. 

Make a PCR master mix as described containing excess amonnt of dNTP and Taq polymerase enzyme. For instance, we fonnd that if mnltiplexing for amplification of promoters of five genes has to be performed at the same time, 0.2 pL 10 pM dNTP and 1.25 U Taq DNA polymerase (Invitrogen) for each gene in the reaction are reqnired. For each gene, consider lOpL of this PCR master mix. 

Polymerase chain reaction (PCR) Master Mix containing Taq DNA polymerase, deoxynncleotide 5 -triphosphate (dNTP), MgCl, and reaction buffers (Promega). 

Setup a PCR reaction starting with 0.05-0.2 pmol of template DNA, 50 pmol of each primer, 10 pL 10X PCR buffer, 10 pL 10X cINTP mix, 0.5 mM MnCl2, 5 U Taq DNA polymerase, and water to a final volume of 100 pL. The manganese solution should be added just prior to the polymerase (see section 2.4, note 1). 

Gold (ABI-Perkin Elmer, Foster City, CA). Too much DNA can lead to PCR artifacts and hence should be avoided. The four deoxynucleotide triphosphates (dNTP) include dATP, dCTP, dGTP, and dTTP. The mixture can be prepared and stored in aliquots at — 80°C. The commercial vendors that sell the Taq DNA polymerase enzyme also provide PCR reaction buffer either with or without Mg2+. The basic constituents of PCR buffer include 100 mM Tris-Cl, pH 8.3 (at room temperature), 500 mM KC1, and other additives in some brands. 

In one report, anti-Taq DNA polymerase antibody was employed to avoid loss of PCR efficiency. The antibody inhibited the Taq polymerase before PCR reagents attained a high temperature, and this procedure is thus called hot-start PCR. In this procedure, the loss of Taq polymerase due to non-specific binding was reduced. This on-chip hot-start PCR resulted in a more consistent and higher yield than that obtained in the PCR chip without hot-start, and even that in the conventional PCR reaction tube (with hot-start) [917],... 

The DNA polymerases of T. littoralis and P. furiosus have been marketed for use in DNA amplification by the polymerase chain reaction (PCR) method as the Vent and pfu DNA polymerases, respectively. These enzymes are more accurate in vitro than the Thermus aquaticus (Taq) DNA polymerase, both in classical fidelity tests [132] and in PCR [133,134]. Indeed, they have an associated 3 to 5 exonuclease activity involved in proof-reading, whereas the Taq polymerase is devoid of such activity. 

Add 1 pL (50-100 ng) of DNA and 1 pL of each primer (4 pM) to PCR tubes and keep on ice. Make up the PCR master mix, adding the 1 pL Taq DNA polymerase last, straight from -20°C. Vortex and briefly pulse the master mix in a benchtop minifuge (see Note 3). Add 22 pL to each reaction, vortex, and pulse briefly again. Overlay reactions with 30 pL of mineral oil. 

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