HotStarTaq Plus 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

Specificity of cPCR and qPCR assays.
Primer set 1184-F/R was tested using cPCR on DNA from 129 isolates of plant- and soil-associated bacteria and fungi (Table 1). The PCR reactions were conducted in a volume of 25 μl that contained 5 μl of reaction buffer (10×), 10 μl of Q-solution (5×), 1 μl of dNTP mix (10 mM), 1 μl of primer 1184-R (10 μM), o1ne μl of primer 1184-R (10 μM), 10 ng of DNA template, and 0.25 μl of HotStarTaq DNA polymerase (5 units/μl). The Q-solution was included to aid the amplification of GC-rich templates (Qiagen, Valencia, CA). Thermal cycling parameters consisted of 5 min at 95°C, followed by 30 to 40 cycles of 45 s at 94°C, 1 min at 60°C, and 1 min at 72°C. The reaction was extended for 10 min at 72°C. Reactions were carried out using a GeneAmp PCR System 2700 (Applied Biosystems). Production of a predicted 666-bp amplicon was considered confirmation of CMN_01184 and C. michiganensis subsp. nebraskensis in a given sample.

The specificity of primer set 1184-qF2/qR2 for qPCR was evaluated using the same bacterial and fungal isolate collection that was used for the cPCR testing (Table 1). The qPCR reactions were conducted using SYBR green PCR master mix (Applied Biosystems). Reactions were conducted in a volume of 40 μl, using 0.8 μl of primer 1184-qF2 (10 μM), 0.8 μl of primer 1184-qR2 (10 μM), DNA template (50 ng), and 20 μl of Power SYBR Green PCR master mix (2×). Thermal cycling parameters were 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 30 s at 61°C, and 30 s at 72°C. Reactions were carried out using an ABI 7500 real-time PCR system (Applied Biosystems). Cycle threshold (CT) values were calculated and were analyzed based on absolute quantification, using StepOnePlus software (Applied Biosystems). CT values less than 35 were considered a positive detection response. All samples were run in duplicate on each of two separate amplification plates.

Sensitivity of cPCR and qPCR assays.
Sensitivity of primer sets 1184-F/R and 1184-qF2/qR2 was evaluated using DNA template from C. michiganensis subsp. nebraskensis CIC251. Sensitivity was tested with two template sets, one represented CFU of C. michiganensis subsp. nebraskensis and the second represented pure, quantified DNA of C. michiganensis subsp. nebraskensis over a range of concentrations. For the CFU templates, C. michiganensis subsp. nebraskensis CIC395 was grown on NBY agar and the cell concentrations were adjusted to 1 × 108 CFU/ml (optical density at 540 nm = 0.1) in 0.85% (wt/vol) NaCl suspension, using a spectrophotometer. Ten-fold serial dilutions were made with the cell suspensions and DNA was extracted from each cell suspension as described above. Using the predicted mass of one C. michiganensis subsp. nebraskensis chromosome, the C. michiganensis subsp. nebraskensis concentration was estimated such that the dilution curve represented one to 1 × 104C. michiganensis subsp. nebraskensis chromosomes per milliliter. For the template set based on pure DNA from C. michiganensis subsp. nebraskensis, a 10-fold serial dilution series was made by diluting a quantified DNA solution to result in concentrations ranging from 3.1 ng to 31 pg per reaction. cPCR and qPCR were conducted with both template sets, as described above. All samples were run in triplicate in three separate reactions.

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Manufacturer protocol

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