| Description |
| |
KlenTaq1 is a 5’-exonuclease deficient Taq polymerase ( an N-terminal deletion of Taq ) with improved fidelity and thermostability. The error rate is about 80% of Taq, and retains at least 85% activity after 1 hour at 95°C.
|
| |
|
| Storage buffer: |
| |
20 mM Tris-HCl, pH 8.55, 222 mM ammonium sulfate, 0.1 mM EDTA 10 mM beta-mercaptoethanol,0.5% Tween 20,0.5 % IGEPAL, 50% (v/v) Glycerol
|
| |
|
| 10X KlenTaq Buffer: |
| |
500 mM Tris-HCl pH 9.,160 mM ammonium sulfate,35 mM MgCl2,1500 μg/ml BSA |
| |
|
| Quality Control: |
| |
No endonucleases, exonucleases and “ nicking activity ” are detected purified enzyme. The enzyme is also DNA free ( no amplification of bacterial gene detected after 35 cycles ).
|
| |
|
| General Protocol: |
| |
Use the following protocol as a starting point and guideline when preparing your reactions. Adjust the reaction size as needed. |
| |
- Select a reaction mixture below based on your type of template. Add the components to a sterile, thin-walled 0.2-ml or 0.5-ml PCR tube either at room temperature or on ice.
|
| |
Note: |
| |
For multiple reactions, it is recommended to prepare a master mix to minimize reagent loss and enable accurate pipetting.
|
| |
|
| |
|
Component
|
Vol./reaction
|
final concentration
|
| 10X KlenTaq Buffer |
5 μl
|
1X
|
| Sense primer (10 μM) |
0.1- 1 μl
|
0.1-1μM
|
| Anti-sense primer (10 μM) |
0.1- 1 μl
|
0.1-1μM
|
| 2.5 mM dNTP each |
8 μl
|
0.2mM
|
| Template DNA |
10pg-500 ng*
|
<10ng/μl
|
| KlenTaq |
0.2 -1μl **
|
1-5u/50μl **
|
| Sterile water |
to 50 μl
|
|
|
| |
* The amount of DNA template varies according to complexity of its sequence. In the case of mammalian DNA, up to 0.5μg is used per reaction. Typical amount of yeast, bacterial, and plasmid DNAs used per reaction are 10ng, 1ng, and 10pg, respectively.
** 0.2 μl = 1.0 unit, which is sufficient for amplifying most small targets ( < 1kb ). For longer targets, use about 1u per kb. |
| |
|
| |
- Mix and overlay with mineral oil, if necessary.
- Perform of PCR amplification as follows:
|
| |
| 1 cycle |
Denature 94°C for 2 min |
| 25-35 cycles |
Denature 94°C for 30 s -1min
Anneal 52-64°C for 30 s -1min
Extend 72°C for 1 min per kb
|
| 1 cycle |
Extend 72°C for 7 min |
| Soak |
4°C indefinite |
|
| |
|
| |
|
| |
|
