Cat. No. / ID: Not Applicable
Taq-B DNA Polymerase is a thermally stable, processive 5ʹ→3ʹ DNA polymerase. The 94 kDa protein possesses an inherent 5ʹ→3ʹ nick-translation moiety and lacks a 3ʹ→5ʹ proofreading function. Taq-B DNA Polymerase is the industry standard for routine 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.
10x PCR Buffer l (cat. no. B7030) contains 100 mM Tris-HCl, 500 mM KCl and 5 mM MgCl2; pH 8.3 at 25°C.
Low-glycerol formulations available.
|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.
Molecular weight: 94 kDa
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-B 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-B DNA Polymerase.
Quality control analysis
Unit characterization assay
Specific activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1x reaction buffer and added to 50 µL reactions containing calf thymus DNA, 25 mM TAPS (pH 9.3), 50 mM KCl, 2.0mM MgCl2, 1 mM DTT, 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.