Cat. No. / ID: P7110L
Taq DSC 2.0 DNA Polymerase provides a novel, nucleic acid-based hot start, enabling high-performance PCR.
The enzyme is supplied in 20 mM Tris-HCl, 100 mM NaCl, 1.0 mM DTT, 0.1 mM EDTA, Stabilizer and 50% glycerol; pH 7.5 at 25°C.
The 10X PCR Buffer l (B7030) contains 100 mM Tris-HCl, 500 mM KCl and 15 mM MgCl2; pH 8.3 at 25°C
OEM by QIAGEN offers customized bulk manufacturing of this enzyme.
Taq DSC 2.0 DNA Polymerase is thermostable, processive 5ʹ→3ʹ DNA polymerase. The 94 kDa protein possesses an inherent 5ʹ→3ʹ nick-translation moiety and lacks a 3ʹ→5ʹ proofreading function. The DSC formulation contains a nucleic acid based hot-start additive designed to sequester the polymerase during reaction setup and during low-temperature cycling reaction phases.
The hot-start polymerase market has been split into two major segments based on the hot-start method: antibody or chemical. Antibody methods are preferred for their rapid reactivation time, minimal cycle times and increased success rates as the weaker association of an antibody with the polymerase yields more units of active polymerase instantly. Previously, chemical methods provided advantages over antibody methods, as complete inactivation allows reaction mixtures to remain inactive for multiple days, despite their longer reactivation, which can be up to 10 minutes. This allows automation and stacking of reactions, something that — until now — has been out of reach of antibody-based methods.
|Specific activity||74,625 U/mg|
|Single-stranded exonuclease||50 U, <5.0% released|
|Double-stranded exonuclease||50 U, <1.0% released|
|Double-stranded endonuclease||50 U, no conversion|
|E. coli DNA contamination||50 U, <10 copies|
Source of recombinant enzyme protein
A recombinant E. coli strain carrying the Taq DNA polymerase gene from the thermophilic organism Thermus aquaticus YT-1.
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 75°C.
Reactions are typical µL.
On ice, prepare each of following master mixes, combine, and place in heated (94°C) thermal cycler.
2x DNA/oligonucleotide master mix:
1.0 µL 10 mM dNTPs
1.0 µL 10 µM Forward Primer
1.0 µL 10 µM Reverse Primer
1.0 µL 500 ng/µL genomic DNA
21 µL Type I Water
2x enzyme/buffer master mix:
5.0 µL 10x PCR Buffer I
0.2 µl 5 U/µL Taq DSC 2.0 DNA Polymerase
19.8 µL Type I Water
|General cycling conditions|
|Step||Initial denaturation||Denaturation:||Annealing||500 bp extension||Final extension|
|Time||3 minutes||30 seconds||30 seconds||30 seconds||5 minutes|
|Cycles||1 cycle||25 cycles||25 cycles||1 cycle||1 cycle|
Taq DNA Polymerase is the original and most commonly used PCR enzyme. Taq excels at amplifying short (<5 kb) sequences from low-complexity template sources and produces robust yields with little or no optimization of reaction conditions.
Consider the following guidelines when designing PCR strategies using Taq DSC 2.0 DNA Polymerase.
Quality control analysis
Specific activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer ([Taq-DSC 2.0]f = 0.01-0.00008µg/µl) and added to 50 µl reactions containing 10 µg calf thymus DNA, 25 mM TAPS (pH 9.3), 50 mM KCl, 2.0 mM MgCl2, 1 mM DTT, 4 mCi/ml 3H-dTTP and 100 µM dNTPs. Reactions were incubated 10 minutes at 75°C, placed on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26).
Protein concentration is determined by OD280 absorbance.
Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.
Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.
Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.
Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.
E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.
This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.