Taq DNA polymerase (Enhancer for high GC template)

Product#: 02-03
$43.00
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Taq DNA polymerase (Enhancer for high GC template)


Description

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)

Products
 
Product ID dNTP Size
02-03_02-013
- 200 units
02-03_02-013-5
- 5x200 units
02-03_02-003
+ 200 units
02-03_02-003-5
+ 5x200 units


General composition of PCR reaction mixture (total 50ul)

Taq DNA polymerase (5 units/ul) *0.25 ul
10 x Standard Buffer (Taq) 5 ul
2.5mM (each) dNTPs 4ul
Template <500ng
Primer 1 0.2~1.0uM (final conc.)
Primer 2 0.2~1.0uM (final conc.)
Sterile distilled water up to 50ul

*Use of excess amount of enzyme is not recommended. 


Storage Conditions

20mM Tris-HCl (pH 8.0), 100mM KCl, 0.1mM EDTA, 1mM DTT, 50% glycerol, 0.5% Tween20, 0.5% Igepal CA-630, Store at -20℃

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℃ 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). 

Reagents Supplied with Enzyme
  1. 10 x Robust Buffer (Taq)
  2. 5 x GC Enhancer 
02-013-5.jpg
Fig.1 SDS-PAGE analysis of Taq DNA polymerase

Cautions for usage of Robust Buffer (Taq) without GC Enhancer

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. 

Protocols for PCR

Examples of PCR coditions without GC Enhancer for the amplification of various sizes of λDNA (Results shown in Fig.2)

02-013-5_result1.jpg

Examples of PCR coditions with GC Enhancer for the amplification of the adenylate cyclaseA gene from Bordetella pertussis(ToHAMA I) genomic DNA (GCcontent 67%) (Results shown in Fig.3) 

02-013-5_result2.jpg

02-013-5_result3.jpg
Fig.3
Effect of the Enhancer on the efficiecy of POR with high GC template (the adenylate cyclase
gene from Bordetella pertussis; 67% GC, 6 kb) 

download.png

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