Taq-B DNA Polymerase is a thermally stable, processive, 5′→ 3′ DNA polymerase. The 94 kDa protein possesses an inherent 5′→ 3′ nick-translation moiety and lacks a 3′→ 5′ proofreading function.
Source of Protein
A recombinant E. coli strain carrying the Taq DNA polymerase gene from the thermophilic organism Thermus Aquaticus YT-1.
20 mM Tris-HCl
100 mM NaCl
1.0 mM DTT
0.1 mM EDTA
pH 7.5 @ 25°C
B7030 (10X PCR Buffer I)
10X PCR Buffer l (B7030)
100 mM Tris-HCl
500 mM KCl
15 mM MgCl2
pH 8.3 @ 25°C
1 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.
Unit Characterization Assay
Specific activity was measured using a 2-fold 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, plunged on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.25-A8.26)
Protein Concentration (OD280) Measurement
A 2.0 µL sample of enzyme was analyzed at OD280 using a Nanodrop ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209) and blanked with product storage solution. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 110,380 and molecular weight of 93,910 Daltons
SDS-Page (Physical Purity Assessment)
2.0 µL of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µL of a 1:100 dilution of the sample. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.
Single-Stranded Exonuclease Activity
A 50 µl reaction containing 10,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 5.0% release of TCA-soluble counts.
Double-Stranded Exonuclease Activity
A 50 µl reaction containing 5,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in less than 1.0% release of TCA-soluble counts.
Double-Stranded Endonuclease Activity
A 50 µL reaction containing 0.5 µg of pBR322 DNA and 10 µL of enzyme solution incubated for 4 hours at 37°C resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis.
E.coli 16S rDNA Contamination Test
Replicate 5uL samples of enzyme solution were 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. The acceptance criterion for the test is the threshold cycle count (Ct) produced by the average of 3 replicate no template control samples. Based on the correlation between the no template control Ct values, and standard curve data, the detection limit of this assay is <10 copies genome/sample.
|Taq-B DNA Polymerase|
|Unit Size||10,000 U|
|SDS||Available on request|
|Storage Temperature||-25 to -15°C|
|Specific Activity||74,625 U/mg|
|SS Exonuclease||50 U <5.0% released|
|DS Exonuclease||50 U <1.0% released|
|DS Endonuclease||50 U = No conversion|
|E.coli DNA Contamination||50 U <10 copies|
* For a detailed summary of assay conditions and data, refer to the Quality Controls Analysis section.
Typical 50 µL Reaction
On ice, prepare each of following master mixes, combine, and place in heated (to 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 DNA Polymerase
19.8 µL Type I Water
General Cycling Conditions
94°C 3 minutes Initial Denaturation
94°C 30 seconds Denaturation
55°C 30 seconds Annealing
68°C 30 seconds 500 bp extension
68°C 5 minutes Final Extension
Taq DNA Polymerase is the original and most commonly used PCR enzyme. Taq excels at amplifying shorter (<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 DNA Polymerase.
Certain applications in which this product can be used may be covered by patents issued and applicable in the United States and abroad. Purchase of this product does not include a license to perform any patented application, therefore it is the sole responsibility of users of this product to determine whether they may be required to engage a license agreement depending upon the particular application in which the product is used.
Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.
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