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 F0934K
KOD -Plus-
KOD-201 200 U 200 reactions
Store at -20°C Contents
[1]Introduction
[2]Components
上海裸照[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 AUSION
All reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe general laboratory precaution and utilize safety 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
等待歌词[ 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 ratio 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 reagent 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
张继科个人资料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 check can be performed by amplifying the β-globin gene (3.6 Kb) and p53 gene (4.0 Kb).
JAPAN CHINA
TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD.
Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140
世纪之战主题曲
tech_osaka@toyobo.jp 2
[ 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.
* 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 DMSO (final conc. 2-5%) might be effective for amplification of GC-rich targets. Decreased PCR fidelity has been confirmed to not take place with DMSO.
-Contaminated RNA (used for cDNA) or genomic DNA inhibits the PCR reaction by chelating Mg2+. PCR should be performed using template DNA containing <100 ng RNA component.
Component Volume Final
Concentration 10x Buffer for KOD -Plus- 5 μl 1x
2mM dNTPs* 5 μl 0.2 mM each
25mM MgSO4 2
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.
美女美女我爱你< 2-step cycle >
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.
有梦不觉夜漫长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.
Example 2. Effect of addition of DMSO for GC-rich targets.
M: 1kb Template: Human genomic DNA Target: TGF-βgene (GC%=70) 2kb Ladder Markers 1: KOD -Plus-, 0% DMSO 2: KOD -Plus-, 2% DMSO 3: KOD -Plus-, 5% DMSO
M 1 2 3
M 123456M
发布评论