This product uses the engineered hot-start Epitech Taq DNA Polymerase, which has enhanced binding ability to DNA templates and significantly improved anti-interference capability. The enzyme retains the 5’→3’ exonuclease activity of Taq enzyme and can be used for probe-based fluorescent quantitative PCR detection. Meanwhile, Taq monoclonal antibody is added to modify the original enzyme, enhancing the blocking effect on the active site of Taq enzyme and improving specificity. Before high-temperature heating, the Taq monoclonal antibody binds to Taq polymerase to inhibit its activity, thereby suppressing non-specific amplification caused by non-specific annealing of primers or primer dimers under low-temperature conditions. When the amplification reaction system is heated to 95°C, the polymerase activity is restored due to the denaturation of Taq monoclonal antibody, so no special inactivation treatment is required, and it can be used under conventional PCR reaction conditions.
With an optimized buffer system, Epitech Taq DNA Polymerase is suitable for PCR reactions of DNA samples treated with bisulfite conversion, and has better amplification efficiency and sensitivity for template DNA containing uracil after bisulfite conversion. Compared with ordinary DNA, DNA after bisulfite conversion is severely damaged, which will affect the performance of PCR reactions. Similarly, for amplifying damaged DNA samples, cytosine deamination occurs spontaneously over a long period of time, and the deamination rate accelerates when the temperature rises, leading to the accumulation of uracil in DNA and free nucleotides. When other correction enzymes are ineffective, Epitech Taq DNA Polymerase can efficiently amplify such damaged DNA templates containing uracil.
Component List
FP1508958 | Component | 250U | 1KU | 5KU | Storage |
FP1508958A | Epitech Taq DNA Polymerase (5U/μL)
| 50μl | 200μl | 1ml | -20℃. Avoid freeze/thaw cycle. |
FP1508958B | 5×Epitech Taq DNA Polymerase Buffer
| 1ml | 4ml | 20ml | -20℃. Avoid freeze/thaw cycle |
Scope of Application
Suitable for PCR reactions of DNA samples treated with bisulfite conversion.
Precautions
In this experiment, the pretreatment process of bisulfite will directly affect the quality of template DNA; the treated template cannot be stored for a long time and should not be kept at -20°C for more than one month.
Other Precautions
1.Excessive template DNA is prone to cause non-specific PCR products.
2.Recommendations for the use of templates: Take mammalian and E. coli genomic DNA as examples:
| Reagent Name | Addition Amount |
|---|
| Mammalian Genomic DNA | 0.1-1 μg |
| E. coli Genomic DNA | 10-100 ng |
Usage Method
Preparation before reaction system preparation:
1. Thaw and mix all solutions required for the reaction at room temperature or 4°C. Then place them on an ice bath or in an ice box. It is recommended to aliquot the reaction solutions for use to avoid repeated freeze-thaw cycles.
2. Prepare the reaction system with reference to the following table. It is recommended to prepare the reaction system on an ice bath or in an ice box.
|
Component
| Addition Volume per Reaction
| Final Concentration
|
|---|
5×Epitech Taq DNA Polymerase Buffer
| 10 μL | 1× |
Epitech Taq DNA Polymerase
| 0.5 μL | 0.05 U/μL |
| dNTPs | 0.5 μL | 0.25 mM |
Primer-probe Mixᵃ
| X μL | —— |
Template
| X μL | —— |
ddH₂O
| To 50 μL | —— |
Final Volume
| 50 μL |
|
Note: a. The amounts of primers, probes, and templates can be adjusted according to actual needs.
3. Gently pipette to mix or vortex slightly, then centrifuge at room temperature for a few seconds to collect the liquid at the bottom of the tube.
4. Place each prepared PCR reaction tube in a PCR instrument and start the PCR reaction.
Reaction Procedure
Fluorescent Quantitative PCR
Step Name
| Temperature
| Time
| Cycles
|
|---|
Pre-denaturation
| 95 °C | 10 min | 1 |
Denaturationᵃ
| 94 °C | 15 s | 40-45 |
Annealing + Extensionᵃ
| 60 °C | 40 s (Collect Fluorescence)
| 40-45 |
a. Reaction conditions can be adjusted according to the primers, probes, and templates.
Experimental Example
In this experiment, the colorectal cancer-related genes SDC2 and TFPI2 were used as target genes for detection, and ACTB was used as an internal reference gene. The detection results are as follows (template concentration: 10 ng/μL):
ZJ26F0535446