KOD Dash DNA Polymerase
Code No：GK1102-250U Storage：-20℃
|
Product component |
Size |
|
KOD Dash DNA Polymerase |
0.1 ml250U (2.5U/μl） |
|
dNTPs |
0.1 ml（10 mM） |
|
MgCl2 |
1ml（20mM） |
|
10×PCR buffer 1 |
1ml |
|
10×PCR buffer 2 |
1ml |
Description:
KOD Dash is a highly efficient DNA polymerase mixture. It composed of two DNA polymerase according to the optimal mixing ratio: (1) KOD DNA Polymerase having 3'→5' exonuclease activity, (2)modified KOD DNA polymerase getting 3'→5' exonuclease activity-deficient mutant by genetic engineering. KOD Dash having excellent PCR features such as high reaction efficiency and extensibility
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 Super PCR amplification efficiency, can be high afficienly for PCR amplification from small amounts template,alsocan be used for PCR of virus / bacteria testing.
Ø Fast synthesis rate, is the fastest polymerase reaction in the PCR reaction, PCR amplification time can be shortened.
Ø Long extension of the enzyme is a mixture of KOD and KOD Mutant enzymes, 15kb fragment of lambda DNA could be amplified.
Ø Fast DNA extension speed . Extension speed is 1Kb/30s, 2x faster than Taq DNA Polymerase!
Basic reaction conditions
|
Components |
Amount |
|
KOD Dash |
2.5 U |
|
10×PCR buffer |
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（pH8.8） 100mM KCl 60mM (NH4)2SO4 12mM MgCl2 1% TritonX-100 100μg/ml BSA 50% Glycerol
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℃
The cycle(3) is proposed to use for Long fragment PCR. Extension speed::1min/kb.