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F0938K

Instruction manual Blend TaqTM and Blend TaqTM -Plus- 2004

Blend TaqTM / Blend TaqTM Plus<Blend TaqTM><Blend TaqTM -Plus->

BTQ-101
BTQ-201

250 U 200 reactions
250 U 200 reactions

Store at -20°C

Contents[1]

Introduction[2]

Components[3]

Quality testing[4]

Primer design[5]

Cloning of PCR products[6]

Protocol
1. Standard reaction setup
2. Cycling conditions[7]

Examples[8]

Troubleshooting[9] Related products[10]

References

CAUSIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe generallaboratory precaution and utilize safety while using this kit.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

1

www.toyobo.co.jp/e/bio[ 1 ] Introduction

Description
Blend TaqTM and Blend TaqTM -Plus- are highly efficient Taq-based DNA polymerases
developed based on the Barns’ method.1) This method uses a DNA polymerase which
lacked 3’5’ exonuclease (proofreading) activity (e.g., Taq DNA polymerase) and a small
amount of an archaeal DNA polymerase with proofreading activity. Because theproofreading activity repairs misincorporated nucleotide bases causing the termination of
the polymerase reaction, PCR with a ‘mixed’ enzyme solution enables highly efficient
amplification.
The enzyme solution of Blend TaqTM -Plus- contains anti-Taq DNA polymerase
antibodies that inhibit polymerase activity, allowing for Hot Start PCR.
Blend TaqTM and Blend TaqTM -Plus- generate dA overhang-ended PCR products.
Therefore, the PCR products can be cloned using a standard TA-cloning method.

Features-This enzyme is effective for the amplification of various targets from small template
amounts. The elongation ability of this enzyme is much greater than that of the normal
Taq DNA polymerase.
-Hot Start technology using anti-Taq DNA polymerase antibodies results in highly efficient
amplification. <Blend TaqTM (Code No. BTQ-101) does not use hot start>-The PCR error ratio of this enzyme is approximately 3-4 times less than that of Taq DNApolymerase.

Fig. 1. The principles of the Barns’ technology.

[ 2 ] Components<Blend TaqTM><Blend TaqTM -Plus->

The provided reagents include the following components for 200 reactions:
Blend TaqTM (2.5 U/µL)
10x PCR Buffer for Blend TaqTM
2 mM dNTPs

100 µL
1.0 mL
1.0 mL

Blend TaqTM -Plus- (2.5 U//µL)*
10x PCR Buffer for Blend TaqTM
2 mM dNTPs

100 µL
1.0 mL
1.0 mL*This enzyme solution contains anti-Taq DNA polymerase antibodies.
JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

1

www.toyobo.co.jp/e/bio[3]

Quality Testing[4]

Primer Design

Quality check was performed by amplifying the human β-globin gene (17.5 kb).

PCR primers should be designed according to general guidelines. For the amplification ofa long target (≥ 6 kb), the melting temperature (Tm) of the primers should be set over 70 °C.

[ 5 ] Cloning of
PCR products

The PCR products of Blend TaqTM and Blend TaqTM -Plus- can be cloned according to a
standard TA-cloning method.

[ 6 ] Protocol

1. Standard reaction setup
The following procedures are designed to be used with the components provided in thiskit. Before preparing the reaction mixture, all components should be completely thawed,except for the enzyme solution.

Component
10x Buffer
2 mM dNTPs*
10 pmol/μL Primer #1
10 pmol/μL Primer #2

Volume
5 µL
5 µL
1 µL
1 µL

Template DNAX µL

PCR grade water
Blend TaqTM (2.5 U/μL ) orBlend TaqTM -Plus (2.5 U/μL
Total reaction volumeY µL
0.5 µL

Final Concentration
1x0.2 mM each
0.2 µM
0.2 µM
Genomic DNA 10-1000 ng/50 µL
Plasmid DNA 1-50 ng/50 µL
cDNA ≤200 ng （RNA equiv.)/50 µL
E. coli cells (small amount)
1.25 U / 50 µL

50 µL* Do not use dNTPs from other kits or companies.
Notes:-Thin-wall tubes are recommended for PCR use. A total reaction volume of 50 µL isrecommended.
-For amplification of a long target (≥ 10 kb), the final concentration of dNTPs should be
0.3-0.4 mM.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

2

www.toyobo.co.jp/e/bio

2. Cycling conditions
The following cycling steps are recommended.

(1) Cycling conditions for < 6kb targets.

< 3-step cycle >Pre-denaturation

94 °C, 2 min.

Denaturation

94 °C, 30 sec.

AnnealingTm-[5-10] oC*, 30 sec.

Extension

72 °C, 1 min./kb

25-35 cycles*Tm value of the primer minus 5°C-10°C(2) Cycling conditions for ≥ 6kb products.
< 2-step cycle >Pre-denaturation

94 °C, 2 min.

Denaturation

94 °C, 30 sec.

Extension

68 °C, 1 min./kb

25-35 cycles

Note: If the Tm value of the primer is under 73 °C, the 3-step cycle is recommended.
Notes:-Extension time should be set at 1min. per 1 kb of target length.
-For colony-direct PCR, the pre-denaturation step should be set for 4 min.

[ 7 ] Examples
Example 1．Comparison of the sensitivity of PCR with a Taq-based PCR
enzyme.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

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Example 2．Colony-direct PCR using E. coli cells.

[ 8 ] Troubleshooting
Symptoms

Cause
Cycling conditions are not
optimal

No PCR product / low yield

Primers are not good
Too much E. coli cells(Colony direct PCR)
Cycling conditions are not
optimalSmearing / extra band(s)Solution
Lower annealing temperature increments to a
maximum of Tm-5 °C.
Increase the number of cycles by 2-5 cycles.
Check the design and/or quality of primers.
Decrease the amount of E. coli cells from colonies.
Decrease the number of cycles by 2-5 cycles.

[ 9 ] Related products
Product name

Package
100 rxns

Code No.
FSK-101

Highly efficient reverse transcriptase
ReverTra AceTM

10,000 U

TRT-101

RNase inhibitor (Recombinant type)2,500 U
200 U x1

SIN-201
LDP-101

200 U x1

KOD-201

200 U x1KFX-101

Highly efficient cDNA synthesis kit
ReverTra AceTM -α-High Speed PCR enzyme

KOD DashHigh fidelity PCR enzyme

KOD -PlusHighly reliable PCR enzyme

KOD FX[ 10 ] References

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

1) Barns WM, Proc. Natl. Acad. Sci. USA, 91: 2216-2220 (1994)CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

4