FastStart™ Taq 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.	Always thaw on ice

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.
Protocol tips
Always thaw on ice

Publication protocol

Endpoint PCR was conducted in a total volume of 20 µL containing 0.4 µM each primer (Table 1), 0.25 mM each dNTP, 1 U or 4 U FastStart Taq DNA polymerase (Roche, Basel, Switzerland) and 1 x FastStart Taq PCR reaction buffer with 2 mM MgCl2. Cycling conditions were at 95°C for 15 min and 40 cycles at 95°C/30 s, 54°C/30 s, 72°C/30 s.
qPCR was performed using 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). 20-µL reactions were conducted in triplicates, containing 0.4 µM of each primer (0.15 µM ACTB bisulfite primer, Table 1), 0.3 µM locus-specific hydrolysis probe (Table 1), 0.25 mM dNTPs, 1 x PCR buffer [35 mM Tris (pH8.4), 50 mM KCl, 6 mM MgCl2], 0.125 x SYBR Green® I (Lonza, Rockland, ME, USA) and varying amounts of FastStart Taq DNA polymerase. Cycling conditions were as follows: 95°C for 15 min initial denaturation, followed by 40 cycles consisting of annealing, extension and denaturation (95°C for 15 s). Annealing and elongation conditions were adjusted for each locus as follows: PITX2 (genomic) locus (56°C for 1 min, 70°C for 30 s), ACTB (genomic) locus (58°C for 1 min, 70°C for 30 s), and ACTB (bisulfite-converted) locus (56°C for 45 s). The amplification under varying thermal cycling conditions was performed as followed: annealing at 56°C for 30 s, 1 min or 2 min and elongation at 70°C for 15 s, 30 s or 1 min, respectively. The threshold level was set to 0.01 and the baseline 3 to 15 for the hydrolysis probe detection. For ACTB bisulfite reactions default threshold level provided by Fast Real-Time PCR System were used.

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Manufacturer protocol

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