KOD DNA Polymerase
Code No：GK1101-250U Storage：-20℃
|
Product component |
Size |
|
KOD DNA Polymerase |
0.1 ml250U (2.5U/μl） |
|
dNTPs |
100μl（10mM） |
|
MgCl2 |
1ml（20mM） |
|
10×PCR buffer 1 |
1ml |
|
10×PCR buffer 2 |
1ml |
Description:
This KOD DNA polymerase is from the hyperthermophilic bacterium, Thermococcus kodakaraensis KOD1, which was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima-ken. It also has high 3’--5’ exonuclease(proofreading) activity, the fidelity is about 50-fold compared to Taq DNA Polymerase.
Unit Definition:
One unit is defined as the amount of enzyme that will catalyze the incorporation of 10nmoles dNTP into acid insoluble form in 30 minutes at 75℃.
Application:
Ø All PCR amplification, especially for the cloning of PCR products;
Ø Make the blunt- end of DNA fragments.
Product features:
Ø High 3’--5’ exonuclease(proofreading) activity, 50-fold higher fidelity than Taq DNA Polymerase, the best choice for PCR amplification.
Ø Fast DNA extension speed . Extension speed is 1Kb/30s, 2x faster than Taq DNA Polymerase!
Ø Excellent heat-resistance. Favorable to deal with high-level structure with specific target fragments such as GC-rich template. At the same time adding to a final concentration of 2-5%DMSO is helpful to improve the amplification effect.
Basic reaction conditions
|
Components |
Amount |
|
KOD DNA Polymerase |
2.5 U |
|
10×PCR buffer 1 |
5 μl |
|
Template DNA |
1-50 ng（plasmid） 10-1000ng（genome） |
|
primer（10uM） |
1 μl |
|
dNTPs |
0.2 mM |
|
Total |
50 μl |
Cycle（1） Cycle（2） Cycle（3）
98℃ 1min 98℃ 1min 98℃ 1min
98℃ 15s 98℃ 15s 98℃ 15s
Tm 5s Tm 30s 68℃ 40s/kb
74℃ 30s/1kb 74℃ 30s/kb
25-35cycles 25-35cycles 25-35cycles
Ø In general the buffer1 is used, the buffer2 is recommended to use when the fragments amplified is greater than 4kb or towing.
Ø It’s not suitable for long-distance PCR. It is suggested to use KOD Dash, another product of our company for long-distance PCR.
12mM Mg2+ has been contained in the buffer.
Ø The cycle(2) could be used when cycle(1) doesn’t work. The cycle(3) could be used when the electrophoresis image is towing. The results usually will improve after changing these conditions.
10×PCR buffer1：
1.2M Tris-HCl（pH8.0） 100mM KCl 60mM (NH4)2SO4 12mM MgCl2 1% TritonX-100 100μg/ml BSA
10×PCR buffer2：
1.2M Tris-HCl（pH8.0） 100mM KCl 60mM (NH4)2SO4 12mM MgCl2 1% TritonX-100 100μg/ml BSA
Storage buffer：
50mM Tris-HCl（pH8.0）0.1mM EDTA50mM KCl 1mM DTT 0.1%Tween-20 0.1% NP-40 50% Glycerol
A few pieces of advice:
Ø Clone PCR products:
The products of this enzyme can be cloned by blunt-ended cloning method. The PCR products should be phosphorylated or amplified with the primers with 5’phosphoryl group when the carrier has been carried out dephosphorylation treatment.
Ø The PCR condition:
The extension speed is 1Kb/30s. The electrophoresis image will towing sometimes if the extension time is too long, so if it tows, you can decrease the extension time or the enzyme amount.
Because the enzyme has excellent heat resistance, it can be denatured at 96℃ or above when the template is easy to form high-level structure or GC rich..
Ø The design of primer:
Longer primer is better compared to Taq DNA polymerase. When the Tm values of the primers are high, the cycle(3) is proposed to use. This enzyme can correct the 3’ end mismatches sometimes if the primers 3’ ends have mismatches.
Ø Program setting:
Extension speed::30s/kb Extension temperature: 74℃