KOD DNA Polymerase
|
包装内容 |
Cat: PC300 |
Cat: PC301 |
Cat: PC302 |
|
KOD -Plus- (1.25 U/μl) |
250U |
250U X4 |
250U X12 |
|
5×KOD BufferⅠ with Mg2+ |
1.0 ml × 1 |
1ml X12 |
1ml X12 |
|
5×KOD BufferⅡ with Mg2+ |
1.0 ml × 1 |
1ml X12 |
1ml X12 |
﹡5×KOD BufferⅡ含有PCR增强剂，可以同时做PCR，择优使用；又可分为含Mg2+和不含Mg2+两种，用户可自选。不特别要求通常提供含Mg2+的。
贮存条件：
-20℃保存
产品特性：
-----扩增准确性是Taq酶的约83倍。
---- 扩增速度是Taq酶的约2倍，pfu酶的约5倍。
-----耐热性比Taq酶更好，在100℃、1h的热处理后仍有约70%的活性，有利于高GC含量样品扩增。
-----产物为平末端
Description
KOD DNA polymerase from Thermococcus kodakaraensis KOD is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase.The enzyme catalyzes the template-dependent polymerization of nucleotides into duplex DNA in the 5’ →3’ direction. The KOD DNA Polymerase also exhibits 3’ →5’exonuclease (proofreading) activity, that enables the polymerase to correct nucleotide incorporation errors. It has no 5’ →3’ exonuclease activity..
Primer Design
-Primers should be 22-34 bases with a melting temperature (Tm) over 60°C. Foramplification 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.
Cloning of PCR products
-KOD generates blunt-end PCR products, due to 3’→5’ exonuclease (proofreading) activity. Therefore, the product can be cloned according to a blunt-end cloning method.
-PCR products of KOD should be purified prior to restriction enzyme treatments. The 3’→5’ exonuclease activity of KOD DNA polymerase remains after the PCR cycles.
Protocol
1. Standard reaction setup
The following procedure is designed for use with the components provided in this kit.
|
Component |
Volume |
Final Concentration |
|
PCR grade water |
Y μl |
|
|
10×KOD Buffer with Mg2+ |
5 μl |
1x |
|
2.5mM dNTPs* |
4 μl |
0.2 mM each |
|
10pmol/μl Primer #1 |
1.5 μl |
0.3 μM |
|
10pmol/μl Primer #2 |
1.5 μl |
0.3 μM Genomic DNA 10-200 ng/50 μl |
|
Template DNA |
X μl |
Plasmid DNA 1-50 ng/50 μl
cDNA ≤ 100 ng （RNA equiv.)/50 μl |
|
KOD-Plus- (1.0 U/μl) |
1 μl |
1.0 U / 50 μl |
|
Total reaction volume |
50 μl |
|
- 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.
2. Cycling conditions
The following cycling steps are recommended.
< 2-step cycle >
Pre-denaturation: 94 °C , 2 min.
Denaturation: 94 °C, 15 sec.
Extension: 68 °C, 10 s～25 s./kb.
Note: If the Tm value of the primer is under 73 °C, the 3-step cycle is recommended.
< 3-step cycle >
Pre-denaturation: 94 °C, 2 min.
Denaturation: 94 °C, 15 sec.
Annealing: Tm-[5-10] °C, 30 sec.
Extension: 68 °C, 10 s～25 s./kb
Note:Tm value of the primer minus 5°C-10°C
Notes:-Extension time should be set to , 10 s～25 s./kb of target length.
|
Step |
Target size |
|
< 500 bp |
500–1000 bp |
1000–3000 bp |
> 3000 bp |
|
1. Polymerase activation |
95°C for 2 min |
95°C for 2 min |
95°C for 2 min |
95°C for 2 min |
|
2. Denature |
95°C for 20 s |
95°C for 20 s |
95°C for 20 s |
95°C for 20 s |
|
3. Annealing |
Lowest Primer Tm°C for 10 s |
|
4. Extension |
70°C for 10 s/kb |
70°C for 15 s/kb |
70°C for 20 s/kb |
70°C for 25 s/kb |
|
Repeat steps 2–4 |
20–40 cycles. For more information see "Cycle number" below |
Examples
Example 1．Effect of Hot Start PCR on the generation of primer dimers.
M 1 2 3 4 5 6 M
Template: Human genomic DNA
1,3,5: 50ng, 2,4,6: 100ng
Target: p53 gene 4kb
M: λ/HindⅢ Marker
1,2: KOD
3,4: A company high fidelity enzyme
5,6: B company high fidelity enzyme
←Primer dimer
Example 2. Effect of addition of DMSO for GC-rich targets.
M 1 2 3
Template: Human genomic DNA
Target: TGF-β gene (GC%=70) 2kb
M: 1kb Ladder Markers
1: KOD, 0% DMSO
2: KOD , 2% DMSO
3: KOD , 5% DMSO
| 
