Biotools DNA Polymerase

Protocol tips

Upstream tips	Protocol tips	Downstream tips
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 conditions.
Conventional PCR was performed in a GeneAmp 9700 thermal cycler (Life Technologies, USA). With culture suspensions and 1:10 dilutions of plant material extract, we used a final volume of 50 μl (45 μl of the mixture and 5 μl of the sample). For DNA extracted from plant material, we used 47 μl of the mixture and 3 μl of the sample. The reaction mixture contained 1 U Taq DNA polymerase (Biotools, Spain) when culture suspensions were analyzed and 2 U Taq DNA polymerase for DNA extracted from plant material (see below), 1 μM each primer, 0.1 mM each deoxynucleoside triphosphate (dNTP), and 3 mM MgCl2. The mixture was subjected to an initial denaturation step of 3 min at 94°C, followed by 40 cycles of 30 s at 94°C, 30 s at 56°C, 45 s at 72°C, and a final extension step of 10 min at 72°C. The amplification products obtained by conventional PCR were visualized by 2% (wt/vol) agarose gel electrophoresis in 0.5× TAE buffer (40 mM Tris, 20 mM sodium acetate [NaOAc], 1 mM EDTA [pH 8], 17.45 M acetic acid [HOAc]) after ethidium bromide staining. A 100-bp molecular weight marker (New England BioLabs, USA) was used. In preliminary experiments, different concentrations of primers (0.5 μM, 0.75 μM, and 1 μM) were tested to optimize reaction conditions.

Real-time PCR assays were performed in a LightCycler 480 thermocycler (Roche, USA) and in a SmartCycler system (Cepheid, USA). With culture suspensions and DNA extracted from plant samples, we used a final volume of 12 μl (9 μl of the mixture and 3 μl of the sample). For 1:10 dilutions of plant material extract (without DNA extraction), we used 20 μl of the mixture and 5 μl of the sample. The reaction mixture contained 2× reaction Master Mix (Biotools, Spain), 0.8 μM each primer, and 0.1 μM TaqMan probe. The amplification conditions were as follows: an initial denaturation step of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 70°C.

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