Taq DNA polymerase Economy (-dNTPs), with Enhancer for high GC
template and Robust Buffer
02-013200 U(5 U/μl)

Storage: Store at -20˚C.
Concentration: 5 units/μl*Note: 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 is used as
template/primer.
Storage Buffer: 20mM Tris-HCl (pH 8.0), 100mM KCl, 0.1mM EDTA, 1mM DTT, 50% glycerol, 0.5%Tween 20, 0.5% Igepal CA-630.
Supplied Reagents:
1) 10 x Robust Buffer (Taq)2) 5 x GC Enhancer
Applications:
1) High-throughput PCR2) Colony PCR3) Incorporation of dUTP, dITP, and fluorescence-labeled nucleotides
4) Primer extension5) Addition of a single nucleotide (adenosine) at the 3’-blunt ends (for cloning into TA vector)
Background: Thermus aquaticus DNA polymerase (Taq DNA polymerase) was expressed in E. coli inlarge quantities and highly purified. The enzyme has thermostable DNA polymerase activity and theMW is 94 kDa. This enzyme kit is especially suitable for PCR reactions with high GC template due toEhancer for high GC templates and Robust Buffer.
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 DNA as a template up to 14
kB (Fig.2).
Cautions for using Robust Buffer (Taq) without GC Enhancer: Robust Buffer induces maximum
enzymatic activity. To avoid production of undesirable smear bands in gel electrophoresis analysis, theoptimal reaction time is recommended as follows: 1) about 5 to 10 seconds / kb elongation time fortemplate up to 8 kb, and about 15 seconds / kb for up to 14 kb; 2) roughly the same elongation time isset with2-step PCR (shuttle PCR) and 3-step PCR; 3) extend the elongation time by short steps when
amplification is not seen. Amplification can be detected more rapidly by adopting 2-step PCR.
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info@bioacademia.co.jp http://www.bioacademia.co.jp/en/General composition of PCR reaction mixture (total 50 μl)

Taq DNA polymerase (5 units/μl)

0.25 μl*

10 x Robust Buffer (Taq)

5 μl

5 x GC Enhancer solution

10 μl

2.5mM (each) dNTPs

TaqDNApolymerase

4 μl

Template<500 ng

Primer 1

0.2～1.0 μM (final conc.)

Primer 2

0.2～1.0 μM (final conc.)

Sterile distilled waterup to 50 μl*Use of excess amount of the enzyme is not recommended.

Fig.1

SDS-PAGE analysis ofTaq DNA polymerase

Protocols for PCR
Examples of PCR coditions without GC Enhancer for the amplification of various sizes of λDNA
(results shown in Fig.2)
2 kb, 4 kb

6 kb

8 kb

94 ﾟ C1 min

94 ﾟ C 1 min

94 ﾟ C 1 min

95 ﾟ C5 sec

95 ﾟ C 5 sec

95 ﾟ C 5 sec

65 ﾟ C 20 sec

25 cycles

65 ﾟ C 1 min

25 cycles

10 kb, 12 kb

65 ﾟ C 1 min 20 sec

25 cycles

14 kb

94 ﾟ C 1 min

94 ﾟ C 1 min

98 ﾟ C 5 sec

98 ﾟ C 5 sec

68 ﾟ C 3 min

30 cycles

68 ﾟ C 4 min

72 ﾟ C 3 min

30 cycles

72 ﾟ C 4 min
M1234567M marker
14 kb
12 kb10 kb
8 kb
6 kb
8kb
8kb
- --4 kb
8kb
- kb
2-BioAcademia,Inc.

-

12 kb

24 kb

36 kb

48 kb

5 10 kb
6 12 kb
7 14 kbFig. 2 PCR products obtained by using Robust Buffer (agarose gel electrophoresis)
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Examples of PCR coditions with GC Enhancer for the amplification of the adenylate cyclaseA genefrom Bordetella pertussis（ToHAMA I) genomic DNA (GCcontent 67%)(Results shown in Fig.3)

98 ﾟ C 2min
98 ﾟ C 5sec
68 ﾟ C 1min

14 cycles

98 ﾟ C 5sec

* decrease 0.5 ﾟ C / cycle

68 ﾟ C * 1min

16 cycles

72 ﾟ C 3minFig.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)

M

123M Marker
1 without GC Enhancer
2 with GC Enhancer
3 NcoI digestion of the PCR product
The adenylate cyclase A gene has a unique NcoI site.
The sizes of the digested fragments corresponded tothose expected from the physical map.
GC Enhancer consists of the mixture of reagents that
decrease a melting point of DNA and stabilize DNA -enzyme
interaction.
Five-time dilution of 5x Enhancer is the maximum
concentration that can be used. Users are recommended touse 10-time dilution and increase the concentrations to
5-time dilution if it is necessary to optimize the PCR
reaction.

BioAcademia,Inc.

Tel. 81-6-6877-2335

Fax. 81-6-6877-2336

info@bioacademia.co.jp http://www.bioacademia.co.jp/en/