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Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140
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1 F0934K
KOD -Plus-
KOD-201 200 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] References
[10] Related products
C AUTION
All reagents in this kit are intended for research purposes only. Not for diagnostic or clinical use. Please observe general laboratory safety precautions while using this kit.
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.
Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140
tech_osaka@toyobo.jp 1
[ 1 ] Introduction
[ 2 ] Components [ 3 ] Quality Testing Description
KOD -Plus- is based on DNA polymerase from the hyperthermophilic Archaeon Thermococcus kodakaraensis KOD11) 2). KOD -Plus- exhibits excellent high PCR fidelity and efficiency. The enzyme solution of KOD -Plus- contains two types of anti-KOD DNA polymerase antibodies that inhibit polymerase and 3’J5’ exonuclease activity, thus allowing for Hot Start PCR3). KOD -Plus- generates blunt-end PCR products, due to 3’J5’ exonuclease (proof-reading) activity.
Features
-Hot Start technology, using anti-KOD DNA polymerase antibodies, results in highly efficient amplification (see Example 1).
-KOD -Plus- enables the following amplifications (maximum): 21 kb from lambda phage DNA, 12 kb from human genomic DNA, and 7 kb from cDNA.
-KOD DNA polymerase has strong 3’J5’ exonuclease (proof-reading) activity. The PCR error rate of KOD -Plus- is approx. 80 times less than Taq DNA polymerase.
Table. 1 PCR fidelity comparison of each PCR enzyme.
*PCR fidelity was based on the mutation frequency of PCR products using a positive-selection base assay with the rpsL gene 4).
This kiy includes the following components for 200 reactions:
KOD -Plus- (1.0 U/μl) * 200 μl × 1
10× Buffer for KOD -Plus- 1.0 ml × 1
25 mM MgSO4 1.0 ml × 1
2 mM dNTPs 1.0 ml × 1
*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize polymerase and 3’J5’ exonuclease activity.
Quality checks are performed by amplifying the β-globin gene (3.6 Kb) and p53 gene (4.0 Kb).
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TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.
Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140
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[ 4 ] Primer Design
[ 5 ] Cloning of
PCR products [ 6 ] Protocol -Primers should be 22-34 bases with a melting temperature (Tm) over 6
0°C. For amplification of a long target, 25-34 bases with high Tm values (≥ 65°C) are recommended. PCR primers should be designed according to the general guidelines.
-KOD-Plus- generates blunt-end PCR products, due to 3’J5’ exonuclease (proof- reading) activity. Therefore, the product can be cloned according to a blunt-end cloning method.
-PCR products of KOD-Plus- should be purified prior to restriction enzyme treatments. The 3’J5’ exonuclease activity of KOD DNA polymerase remains after the PCR cycles.
1. Standard reaction setup
The following procedure is designed for use with the components provided in this kit. Before preparing mixture, all components should be completely thawed, except for the enzyme solution.离线
* Do not use dNTPs from other kits or companies.
Notes:
-For PCR reactions, thin-wall tubes are recommended. A total reaction volume of 50 μl is also recommended.
-The addition of dimethyl sulfoxide (DMSO; final conc. 2-5%) might be effective for amplification of GC-rich targets. No decrease in PCR fidelity has been observed using DMSO.
-Contaminating RNA inhibits the PCR reaction by chelating Mg2+. PCR should be performed using template DNA containing <100 ng of RNA.
Component Volume Final卡玛斯大货车
Concentration 10x Buffer for KOD -Plus- 5 μl 1x
2mM dNTPs* 5 μl 0.2 mM each
25mM MgSO4 2
强捻纱μl 1.0
mM
10pmol/μl Primer #1 1.5 μl 0.3 μM
10pmol/μl Primer #2 1.5 μl 0.3 μM
Template DNA X μl
Genomic DNA 10-200 ng/50 μl
Plasmid DNA 1-50 ng/50 μl
cDNA ≤ 100 ng （RNA equiv.)/50 μl PCR grade water Y μl
KOD-Plus- (1.0 U/μl) 1
μl 1.0 U / 50 μl
Total reaction volume 50 μl
JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.
Tel(81)-6-6348-3888 Tel(86)-21-58794900.bo.co.jp/e/bio
tech_osaka@toyobo.jp
3
2. Cycling conditions
The following cycling steps are recommended.
Note : If the Tm value of the primer is under 73 °C, the 3-step cycle is recommended.
*Tm value of the primer minus 5°C-10°C
Notes:
-Extension time should be set to 1min per 1 kb of target length.
< 2-step cycle >
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Pre-denaturation: 94 °C , 2 min. Denaturation: 94 °C, 15 sec. Extension:
68 °C, 1 min./kb
< 3-step cycle >
Pre-denaturation: 94 °C, 2 min. Denaturation: 94 °C, 15 sec.
Annealing: Tm-[5-10]
o
C*, 30 sec.Extension:
68 °C, 1 min./kb
25-35 cycles
JAPAN CHINA TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.
Tel(81)-6-6348-3888 Tel(86)-21-58794900.bo.co.jp/e/bio
tech_osaka@toyobo.jp
4
[ 7 ] Examples
Example 1．Effect of Hot Start PCR on the generation of primer dimers.
The P53 gene (4 kb) was amplified by KOD-Plus-Ver.2 and high fidelity PCR enzymes from other companies using human genomic DNA as the template. KOD -Plus- successfully amplified the target genes without generating primer-dimmers.
M 1 2 3 4 5 6 M
高校实行什么的
校长负责制Example 2. Effect of addition of DMSO for GC-rich targets.金惠敬
The TGF-b gene (2kb, GC%=70) was amplified using KOD-Plus-Ver.2 with or without DMSO. The addition of 2-3 % DMSO permitted effective amplification of the target.
M 1 2
3
Template: Human genomic DNA 1,3,5: 50ng, 2,4,6: 100ng Target: p53 gene 4kb M: l/Hin d III Marker 1,2: KOD -Plus-
3,4: A company high fidelity enzyme 5,6: B company high fidelity enzyme
Primer dimer
Template: Human genomic DNA Target: TGF-β gene (GC%=70) 2kb M: 1kb Ladder Markers 1: KOD -Plus-, 0% DMSO 2: KOD -Plus-, 2% DMSO 3: KOD -Plus-, 5% DMSO
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