GoTaq® DNA Polymerase

Protocol tips

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Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp

Publication protocol

PCR amplification
A 135 bp artificial hairpin DNA template was amplified with 2 U of SD or GoTaq DNA polymerase. Assay reactions (25 µl) contained: 1× GoTaq reaction buffer, 3 mM MgCl2, 0.2 mM dNTPs (each), 0.1 ng template DNA, 0.2 µM primers H1 and H2 (each). Thermocycling conditions were: preheating 94°C for 1 min, followed by 15 cycles of 94°C (60 s), 64°C (20 s), 72°C (20 s).

A 1.3 kb fragment of the Mycobacterium tuberculosis genome was amplified with 1.25, 2.5, or 5 U of SD or GoTaq DNA polymerase. Assay reactions (25 µl) contained: 1× GoTaq reaction buffer, 3 mM MgCl2, 0.2 mM dNTPs (each), 5 ng Mycobacterium tuberculosis gDNA as a template, 0.2 µM primers Mtu1 and Mtu2 (each). Thermocycl ing conditions were: preheating 92°C for 2 min, followed by 30 cycles of 92°C (20 s), 60°C (30 s), 68°C (2 min).

An 8 kb fragment of λ DNA was amplified with 2.5, 5, 10, or 15 U of SD polymerase, GoTaq polymerase (bothin GoTaq buffer), MyTaq polymerase in GoTaq buffer or MyTaq buffer; rTaq polymerase in GoTaq buffer or Encyclo buffer. Assay reactions (50 µl) contained: 5 ng λ DNA template, 0.25 mM dNTPs (each), 10 pmol (0.2 µM) of each primer (λ1 and λ2), 1× PCR buffer, and 3 mM MgCl2 (GoTaq and MyTaq buffers) or 3.5 mM MgCl2 (Encyclo buffer). Thermocycling conditions were: preheating at 92°C for 2 min, followed by 25 cycles of 92°C (30 s), 60°C (30 s), and 68°C (2 min 40 s; 20 s/kb).

Long-distance amplification of a 17.5 kb fragment of the human b-globin gene was performed with 2.5 U of SD polymerase for 35 cycles: 92°C (25 s), 66.5°C (1 min), 69°C (9 min); initial preheating 45 s at 92°C. Assay reactions (25 µl) contained: 100 ng of human gDNA (New England Biolabs) as template, 0.2 mM dNTPs (each), 5 pmol (0.2 µM) of each primer (HG1 and HG2), 1× SD buffer (Bioron), and 2.75 mM MgCl2.

PCDR and PCR amplification
A murine cDNA library was used as a template, and primers F1, F2, F3, R3, R2, and R1 (Supplementary Table SS) were designed to specifically amplify murine G3PDH cDNA. The relative position of the primers is shown in Figure 3A, the sequence of the amplified DNA, with the positions of the primers indicated, is given in Supplementary Figure S1. Assay reactions (50 µl) contained: 1× GoTaq buffer; 3 mM MgCl2; 0.375 mM dNTPs (each); 20 pmol (0.4 µM) inner primers F3 and R3 (each) and 10 pmol (0.2 µM) outer primers F1 and R1; 0.05 ng of the murine cDNA library as a template. PCR assays contained two primers (F3, R3), and PCDR assays contained four primers (F1, F3, R1, R3). The reactions were carried out with 5, 10, 20, or 40 U of SD polymerase, or with 5 or 10 U of GoTaq polymerase. Thermocycling conditions were: preheating 92°C (1 min 30 s), followed by 20 cycles of 92°C (30 s) and 65°C (1 min).

qPCDR and qPCR amplification
Real-time amplifications of murine G3PDH cDNA sequence were carried out with AmpliFluor primer AF3 (Syntol JSC) (Supplementary Table S1). The AmpliFluor primer is similar to inner primer F3 but includes a hairpin structure with a quencher (BHQ2) and a fluorescent reporter (HEX) at the 5′ end. A quantification cycle (Cq) is determined for each well with Bio-Rad (Hercules, CA) CFX Manager 3.0 by regression analysis. This method is based on the fit of Richards—equation to real-time PCR data by nonlinear regression in order to obtain the best fit estimators of reaction parameters (13). The efficiency of each reaction variant was estimated by standard curve (dilution series of murine cDNA library from 10 to 0.001 pg per reaction). The log of each concentration in the dilution series (x-axis) was plotted against the Cq value for that concentration (y-axis). Then efficiency was determined by the following equation:

Efficiency = 10(−1/slope) −1

Assay reactions (25 µl) contained: 5 U of SD HotStart or GoTaq HotStart DNA polymerase; 1× GoTaq buffer for GoTaq polymerase or 1× SD buffer for SD polymerase; 2.75 mM MgCl2; 0.25 mM dNTPs (each); 0.2 µM inner primers AF3 and R3 (each), 0.1 µM outer primers F2 and R2 (each), and 0.05 µM outer primers F1 and R1 (each); 10, 1, 0.1, 0.01, or 0.001 pg of the murine cDNA library as template.

PCR assays contained two primers, AF3 and R3. PCDR assays contained two inner primers (AF3, R3) and two (F2, R2) or four (F2, R2, F1, R1) outer primers.

Amplifications were carried out using a Bio-Rad CFX96 PCR machine with the following protocol: initial preheating 92°C 2 min, followed by 45 cycles of 92°C (15 s), 66°C (40 s).

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