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Reagents for Genetic Engineerring

Taq DNA polymerase Economy (+dNTPs), with Enhancer for High GC template and Robust buffer

Background

Thermus aquaticus DNA polymerase (Taq DNA polymerase) was expressed in E. coli in large quantities and highly purified. The enzyme has thermostable DNA polymerase activity and the MW is 94 kDa. This enzyme kit is especially suitable for PCR reactions with high GC template due to Ehancer for high GC templates and Robust buffer.

Application

1) High-throughput PCR
2) Colony PCR
3) Incorporation of dUTP, dITP, and fluorescence-labeled nucleotides
4) Primer extension
5) Addition of a single nucleotide (adenosine) at the 3’-blunt ends for cloning into TA vector.

*Use of excess amount is not recommended
General composition of PCR reaction mixture (total 50 µl)
Taq DNA polymerase (5 units/ul) 0.25 µl*
10 x Robust Buffer (Taq) 5 µl
2.5mM (each) dNTPs   4 µl
Template <500 ng
Primer 1 0.2 ~ 1.0 M (final conc.)
Primer 2 0.2 ~ 1.0 M (final conc.)
Sterile distilled water up to 50 µl

< Concentration >
5 units/ul, where one unit is defined as the amount of enzyme that can incorporate 10 nmols of total dNTPs into an acid-insoluble material in 30 minutes at 74 degrees when activated salmon sperm DNA was used as template/primer.

< Quality Assurance >
Greater than 95% purity as determined by SDS-PAGE (CBB staining) (Fig.1) The absence of endonucleases and exonucleases was confirmed.

< PCR Test >
Good amplification result was obtained in PCR reaction using λDNA as a template up to 14 kB (Fig.2).

< Fig1. SDS-PAGE of Taq DNA polymerase >
1. 10 x Robust Buffer (Taq)
2. 5 x GC Enhancer
3. 2.5mM(each) dNTPs

< Fig2. PCR products obtained by using Robust buffer (agarose gel electrophoresis) >
Examples of PCR coditions for the amplification of various sizes of λDNA

< Fig3. Effect of the Enhancer on the efficiecy of POR with high GC template (the adenylate cyclase gene from Bordetella pertussis; 67% GC, 6 kb) >
Examples of PCR coditions with GC Enhancer for the amplification of the adenylate cyclaseA gene from Bordetella pertussis(ToHAMA I) genomic DNA (GCcontent 67%)

NOTE : Cautions for usage of Robust Buffer (Taq ) Robust Buffer induces maximum enzymatic activity. Therefore, cares should be taken to avoid production of undesirable smear bands in gel electrophoresis analysis by longer than optimal reaction time. We recommend about 5 to 10 seconds / kb elongation time for template up to 8 kb, and about 15 seconds / kb for up to 14 kb. We will recommend roughly the same elongation time to be set with 2-step PCR (shuttle PCR) and 3-step PCR. Extend the elongation time by short steps when amplification is not seen. The results of your experiments can be observed more rapidly by adopting 2-step PCR.

Product List

Product Name Cat# Quantity Price

Taq DNA polymerase Economy (+dNTPs) with Enhancer for High GC template and Robust buffer

BAM-02-003-EX 200UNIT

¥ 5,200
$ 70
€ 52

Taq DNA polymerase Economy (+dNTPs) with Enhancer for High GC template and Robust buffer

BAM-02-003-5EX 5*200UNIT

¥ 20,500
$ 274
€ 205

Taq DNA polymerase Economy (-dNTPs) with Enhancer for High GC template and Robust buffer

BAM-02-013-EX 200UNIT

¥ 4,300
$ 58
€ 43

Taq DNA polymerase Economy (-dNTPs) with Enhancer for High GC template and Robust buffer

BAM-02-013-5EX 5*200UNIT

¥ 17,200
$ 230
€ 172

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.