Taq DSC 2.0 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. The DSC formulation contains a novel, nucleic-acid based hot-start additive designed to sequester the polymerase during reaction setup and during low-temperature cycling reaction phases.
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.
|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.
QUALITY CONTROL ANALYSIS
Unit Characterization Assay
Specific activity was measured using a 2-fold 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, 2.0mM MgCl2, 1 mM DTT, 4mCi/mL 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.
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 DSC 2.0 DNA Polymerase
19.8 µL Type I Water
|General Cycling Conditions|
|94°C||3 minutes||Initial Denaturation|
|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 DSC 2.0 DNA Polymerase.
- DNA Template: Although extensive purification of PCR templates is typically not necessary, care should be taken with crude or partially purified DNA sources as handling and chemical agents can adversely affect the PCR process. Exposure to short-wave UV light or other DNA damaging agents should be avoided, as should high ionic strength, detergents such as SDS, loading dyes and phenol. In order to prevent contamination from previous PCR reactions, consider setting up reactions in a positive-pressure hood and with aerosol barrier pipet tips. In a typical 25 cycle PCR, 104 copies of target sequence will yield reproducible amplification product. This corresponds to roughly 0.1-1 ng/ml (final concentration) of plasmid DNA, and 1-10 µg/ml of genomic DNA. The use of lower DNA concentrations typically produces less non-specific product, while higher concentrations can allow for fewer cycles and lower mutation rates.
- Primer Design: Ideally, oligonucleotide primers are 15-30 bases in length, nearly 50% G+C, and have equal (+/- 3°C) annealing temperatures. The use of software to detect self-complementary or hairpin-prone regions is advised and is offered as a service by some synthesis providers. Note that although the 5′-terminus of the primer may contain untemplated sequence, the 3′ end must match perfectly. Typical oligonucleotide concentration in the reaction is 0.1-0.5 µM.
- Magnesium: Magnesium is a critical component of the PCR reaction though its concentration can be modulated to promote various effects. Generally, 1.5-2.0 mM Mg2+ is targeted, but higher concentrations (up to 5 mM) may be used to stimulate the yield of reactions at the expense of fidelity. The converse is also true &ndsh; lower magnesium concentrations will promote higher-fidelity products with a lower overall amplification yield. Note that certain reaction components, in particular template DNA and oligonucleotides, may contribute chelating agents to the reaction which could lower the effective magnesium concentration and starve the reaction.
- dNTPs: Generally, a final concentration of 100-200 µM dNTPs is employed, though higher concentrations may stimulate yields (particularly with longer targets) and lower may offer increases in fidelity. Taq DSC 2.0 DNA Polymerase can also incorporate and read through deoxy Uridine and Inosine, two analogs used in certain applications.
- Taq DSC 2.0 Polymerase: 1 unit/50 µL reaction (20 U/mL) is typical, though additional enzyme may be added to stimulate yields. Taq DSC 2.0 DNA Polymerase extends a DNA template at approximately 1-2000 nucleotides/minute, so it is recommended that 30-60 seconds of extension time be provided per kb, per cycle. Appropriate extension temperatures range from 68-72°C. Because Taq DSC 2.0 DNA Polymerase exploits the natural affinity of a DNA polymerase for a duplex DNA fragment to promote its hot-start function, it does not require an extensive initial denaturation step to activate the 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.