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.

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.


Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

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.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing PCR Hot start PCR - Bacterial DNA using HotStarTaq Plus DNA Polymerase from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.