PRODUCT DESCRIPTION产品描述
Taq-B DNA Polymerase is a thermally stable,processive, 5′→3′DNA polymerase. The 94 kDa proteinpossesses an inherent 5′→3′nick-translationmoiety and lacks a 3′→5′proofreadingfunction.
Source of Protein蛋白来源
A recombinant E. coli strain carrying theTaq DNA polymerase gene from the thermophilic organism Thermus Aquaticus YT-1.
Supplied in
20 mM Tris-HCl
100 mM NaCl
1.0 mM DTT
0.1 mM EDTA
Stabilizer
50% glycerol
pH 7.5 @ 25°C
Supplied With
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
Unit Definition单位定义
1 unit is defined as the amount of enzymethat will incorporate 10 nmol of dNTP into acid-insoluble material in 30minutes at 75°C.
PRODUCT INFORMATION产品信息
Taq-B DNA Polymerase
Part Number P7250L, P7250F
Concentration 5,000 U/ml
Unit Size 10,000 U, 1540U
SDS Available on request
PRODUCT SPECIFICATION*产品特性
Storage Temperature -25 to -15°C
Test Specification
Purity (SDS-PAGE) >99%
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 50U <10 copies
*For a detailed summary of assay conditionsand data, refer to the Quality Controls Analysis section.
USAGE INSTRUCTIONS使用说明
Typical 50 μL Reaction
On ice, prepare each of following mastermixes, 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 InitialDenaturation
25 Cycles:
94°C 30 seconds Denaturation
55°C 30 seconds Annealing
68°C 30 seconds 500 bp extension
68°C 5 minutes Final Extension
NOTES
Taq DNA Polymerase is the original and mostcommonly used PCR enzyme. Taq excels at amplifying shorter (<5 kb) sequencesfrom low-complexity template sources and produces robust yields with little orno optimization of reaction conditions. Consider the following guidelines whendesigning PCR strategies using Taq DNA Polymerase.
DNA Template: Although extensivepurification of PCR templates is typically not necessary, care should be takenwith crude or partially purified DNA sources as handling and chemical agentscan adversely affect the PCR process. Exposure to short-wave UV light or otherDNA damaging agents should be avoided, as should high ionic strength, detergentssuch as SDS, loading dyes and phenol. In order to prevent contamination fromprevious PCR reactions, consider setting up reactions in a positive-pressurehood and with aerosol barrier pipet tips. In a typical 25 cycle PCR, 104 copiesof target sequence will yield reproducible amplification product. Thiscorresponds to roughly 0.1-1 ng/ml (final concentration) of plasmid DNA, and1-10 μg/ml of genomic DNA. The use of lower DNA concentrations typicallyproduces less non-specific product, while higher concentrations can allow forfewer cycles and lower mutation rates.
Primer Design: IIdeally, oligonucleotideprimers are 15-30 bases in length, nearly 50% G+C, and have equal (+/- 3°C) annealing temperatures. The useof software to detect self-complementary or hairpin-prone regions is advisedand is offered as a service by some synthesis providers. Note that although the5′-terminus ofthe primer may contain untemplated sequence, the 3&prime end must matchperfectly. Typical oligonucleotide concentration in the reaction is 0.1-0.5 μM.
Magnesium: Magnesium is a criticalcomponent of the PCR reaction though its concentration can be modulated topromote various effects. Generally, 1.5-2.0 mM Mg2+ is targeted, buthigher concentrations (up to 5 mM) may be used to stimulate the yield ofreactions at the expense of fidelity. The converse is also true – lower magnesium concentrationswill promote higher- fidelity products with a lower overall amplificationyield. Note that certain reaction components, in particular template DNA andoligonucleotides, may contribute chelating agents to the reaction which couldlower the effective magnesium concentration and starve the reaction.
dNTPs: Generally, a final concentration of100-200 μM dNTPs is employed, though higher concentrations may stimulate yields(particularly with longer targets) and lower may offer increases in fidelity.Taq DNA Polymerase can also incorporate and read through deoxy Uridine andInosine, two analogs used in certain applications.
Taq Polymerase: 1 unit/50 μL reaction (20U/mL) is typical, though additional enzyme may be added to stimulate yields.Taq DNA Polymerase extends a DNA template at approximately 2000nucleotides/minute, so it is recommended that 45-60 seconds of extension timebe provided per cycle. Appropriate extension temperatures range from 66-72°C.
Limitations of Use适用范围
This product was developed, manufactured,and sold for in vitro use only. The product is not suitable for administrationto humans or animals.
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