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Figure 4 | BMC Biotechnology

Figure 4

From: Signal and noise in bridging PCR

Figure 4

Stoffel fragment of Taq yields a S/N of 100 The Stoffel fragment of Taq polymerase was used in BPCR. The gel was 1% agarose containing ethidium bromide. The fluorescent image of the gel was acquired and processed with a Macintosh computer, a video camera and the software Scion Image 1.59 (from NIH). The gel image intensity was inverted, and the background was subtracted (horizontal 1D). The band intensity of the 328 bp PCR product was recorded. The intensity profile curve of the 328 bp band is aligned and plotted below the gel lanes. The relative area under each peak is given below the peak. Area values are equalized to Lane 10, whose area value is set as 1.0. Lane 1, DNA size markers: a mixture of three separate PCR products: 235 bp (with primers b1 and M13 revI and template pBS(-)), 495 bp (primers M13 pIII and M13 revI and template Phagescript) and 736 bp (primers b1 and b2 and template pBS (-)). Lanes 2, size markers of Lambda DNA, BstE II-digested, 75 ng/lane. Lanes 3, 4, 5 and 6, PCR with intact source template. Each lane had 5 μl PCR product. Lanes 7, 8, 9, 10 and 11, PCR with source template cut with PvuII. Each lane had 5 μl PCR product. The concentrations of source template of lanes 3 and 7 are adjusted to be equal, 1 × 10-16 mol/μl PCR solution. Lanes 4, 5 and 6 are serial template dilutions of lane 3. Lanes 8, 9, 10 and 11 are serial template dilutions of lane 7.

BPCR was carried out with the Stoffel fragment of Taq polymerase and primers b1 and M13 pV. (See Figure 2 for location of primers and Table 1 for their sequence.) The cut or intact template was made by PCR with the Stoffel fragment, primers b1 and Jp and a plasmid preparation of pBS(-). The other template (reference template) was ssDNA from Phagescript particles. The 1 × PCR buffer contains 10 mM KCl, 10 mM Tris-HCl (pH 8.3 at 25°C) and 2.5 mM MgCl2.

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