HotStarTaq Plus DNA Polymerase

PCR Hot start PCR - Bacterial DNA

PCR Hot start PCR - Bacterial DNA
HotStarTaq Plus DNA Polymerase from Qiagen

Protocol tips

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

Publication protocol

Taq polymerases

The following DNA polymerases from commercial vendors designed for qPCR were used in the experiments reported here: Amplitaq Gold (ABI, CA; Roche lot # J02913); Platinum Taq (Invitrogen, CA; cat # 10966–026; lot 1169610); Platinum HiFi Taq (Invitrogen, CA; cat# 11304–011; lot# 1267490); HotStar Taq (Qiagen, CA; Mat # 1007837; lot # 124125007); JumpStart Taq (Sigma, MO; cat # D-6558; lot # 71K9029). Two polymerases not designed specifically for qPCR (no anti-Taq antibody) were also tested: Amplitaq DNA polymerase (ABI, CA; Roche lot # C00622); Qiagen Taq DNA polymerase (Qiagen, CA; Cat # 201205, lot # 127132149. Additional development studies were carried out with a 2x FastStart Universal SYBR Green I Master Mix (Roche, IN; cat # 04913922001).

PCR reaction buffer and cycling conditions

A common master mix was prepared to test all Taq polymerases. Final concentrations of components in the reaction were: 20 mM TRIS (pH 8.2); 50 mM KCl; 3 mM MgCl2; 375 uM each dNTP; 1% DMSO; 5% glycerin; and 20,000-fold diluted SYBR Green I (Molecular Probes, Invitrogen, Carlsbad CA). Similar results were obtained using the ABI Gold Taq PCR Core Components (ABI, cat# 4304886). PCR reactions were carried out in a final volume of 10 ul on Biorad's Chromo4 Four Color Real Time PCR Detector with Gradient DNA Engine Thermocycler. PCR reactions were initiated with a 94°C heating step for 10 min. Cycling was then carried out with melting at 94°C, 10 sec; annealing at 60°C, 10 sec; extension at 72°C, 20 sec. After 35 or 40 cycles, dissociation analysis was carried out from 60°C to 94°C, with ramping at 0.5°C per minute.

Dilutions of Taq polymerases

Stock solutions of Taq polymerase at 5 U/ul were diluted in 2- or 3-fold increments in 2 ng/ul linear acrylamide. 2.5 ul of each dilution were added to 2.5 ul of 4x mastermix with 2.5 ul oligonucleotide primers (300 nM final each primer) and 2.5 ul of sample or H2O.

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HotStarTaq Plus DNA Polymerase from Qiagen has not yet been reviewed for this experiment

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4 years ago

Author: Qatar

When should I add the polymerase for hot start PCR?

I keep getting a non-specific band in PCR so I would like to try a hot start PCR manually. How should I prepare the mix and at which step should I be adding the polymerase?


4 years ago

Author: Janina Reinhardt Switzerland

Hot start PCR using phusion green DNA polymerase

I am currently performing hot start PCR using phusion green hot start DNA polymerase in order to amplify a 8.8kb insert. However, I cannot get any amplified product at all. I have tried changing some parameters but I am not sure what to do. Any help?

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Check out relevant papers found by Labettor's AI that are relevant for performing PCR Hot start PCR - Bacterial DNA using HotStarTaq Plus DNA Polymerase from Qiagen.

Paper title
Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria
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Manufacturer protocol

Download the product protocol from Qiagen for HotStarTaq Plus DNA Polymerase below.

Download PDF Download manufacturer protocol


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