Klein Taq
| Catalog # | Pack size | Price, Euro | SHOP |
| 118002 | 200 u | 75,00 | Add to cart |
| 118010 | 1000 u | 299,00 | Add to cart |
Datasheet: Klein Taq DNA Polymerase
Deutsches Datenblatt: Klein Taq DNA Polymerase
Description:
KleinTaq is a derivative of Taq DNA polymerase. It is a 5'-exo-minus N-terminally truncated Thermus aquaticus DNA polymerase. As expressed from a gene construct in E.coli, translation initiates at Met236, bypassing the 5'-3' exonuclease domain of the DNA polymerase-encoding gene.
The enzyme reveals a highly active and even more heat-stable DNA polymerase activity in comparison with Taq DNA polymerase. The optimal range of Mg2+ concentration for KleinTaq is broader than for the majority of thermostable polymerases. This feature allows to optimize reaction conditions easier then for other polymerases. Repeated exposure to 98°C does not diminish the enzyme activity. Significant activity remains even after exposure to 99°C.
The mutation rate during polymerization is twofold lower for KleinTaq in comparison with full-length Taq DNA polymerase. KleinTaq has a very low background ability to extend a mismatched 3'-oligonucleotide end making it suitable for mutation analysis with mutation-specific oligonucleotides.
Concentration:
5-10000 units/ml
Unit definition:
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTPs into acid-insoluble form in 30 min at 72°C.
Storage buffer:
10 mM K-phosphate pH 7,0, 100 mM NaCl, 0,5 mM EDTA, 1 mM DTT, 0,01% Tween 20, 50% glycerol
Storage:
-20oC
Reaction buffer 10x incomplete:
160 mM (NH4)2SO4; 670 mM Tris-HCl pH (ph 8,8) ; 0,1% Tween-20
Reaction buffer 10x complete:
160 mM (NH4)2SO4; 670 mM Tris-HCl pH (ph 8,8) ; 0,1% Tween-20, 25 mM MgCl2
Reaction buffer 10x complete II KCl:
500 mM KCl; 100 mM Tris-HCl pH (ph 8,8) ; 0,1% Tween-20, 15 mM MgCl2
Plus one tube MgCl2 (100mM)
Quality control:
Activity, SDS-PAGE purity, absence of endonucleases/ nickases and exonucleases
Klein Taq protocol for ~ 2000 bp amplification
for 50 µl volume:
| DNA template | 10 - 15 ng plasmid DNA | |
| Primer | 0.1 - 1 µM final concentration | |
| dNTPs | 0.2 mM each (final concentration) | |
| KleinTaq | 2-12 units in 50 µl | |
| 10 x Bioron reaction buffer complete (with MgCl2) | 5 µl | MgCl2 is final 2.5 mM |
| Add H2O up to 50 µl |
Thermocycling:
The number of cycles will depend on the amount of template DNA. In most cases 25 cycles are sufficient. For low copy number genes or rare DNAs, 30-35 cycles is recommended. The amplification parameters will vary depending on the primers, amplicon size and the thermal cycler used. It may be necessary to optimize the system. For 5 - 10 kb amplicon increase the extension time to 6-10 min.
For example: Thermal Cycler 480 (Perkin-Elmer-Cetus); amplicon size 500 - 2000 bp, template 10 ng plasmid DNA .
The reaction proceeded in 25 cycles:
94°C 30 - 45 s
58 - 61°C 30 s
72°C 100 s
