Animal Tissue Direct PCR Kit (with Dye)
MCE Animal Tissue Direct PCR Kit (with Dye) is specifically designed for direct amplification from a variety of animal tissue samples. It enables rapid release of genomic DNA from animal tissues (such as insect legs/wings, mouse tails, ears, toes, skin, and internal organs) in a single step, allowing the lysate to be used directly for downstream PCR amplification without the need for conventional DNA extraction and purification procedures, thereby significantly simplifying the workflow.
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Storage :
Lysis Buffer: 4°C, 1 year. If not be used for an extended period, it is recommended to aliquot it for storage. Proteases, Direct PCR Master Mix (2×, with Dye), PCR Enhancer (5×): -20°C, 1 year. Avoid repeated freeze-thaw cycles.
- Manual (5109KB)
- COA
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
Description & Advantages
MCE Animal Tissue Direct PCR Kit (with Dye) is specifically designed for direct amplification from a variety of animal tissue samples. It enables rapid release of genomic DNA from animal tissues (such as insect legs/wings, mouse tails, ears, toes, skin, and internal organs) in a single step, allowing the lysate to be used directly for downstream PCR amplification without the need for conventional DNA extraction and purification procedures, thereby significantly simplifying the workflow.
The kit is supplied with a ready-to-use PCR master mix containing all components required for amplification, including Taq DNA polymerase, dNTPs, optimized buffer system, and tracking dyes. Only primers and template need to be added to initiate the PCR reaction. The amplified products can be directly analyzed by agarose gel electrophoresis without the addition of loading buffer, effectively reducing operational complexity and the risk of contamination. This kit is suitable for applications such as animal genotyping and transgenic identification.
Features of MCE Animal Tissue Direct PCR Kit (with Dye):
1. Fast and Convenient: Tissue samples do not require cutting or grinding. Genomic DNA can be prepared in a single step, with template preparation completed in as little as 10 min and PCR amplification in approximately 50 min.
2. Low Sample Requirement: Only a small amount of tissue is required for effective lysis.
3. High Specificity and Efficiency: Optimized Taq polymerase system with strong template affinity and high amplification specificity, ideal for genotyping and transgenic analysis.
4. User-Friendly Operation: The master mix contains tracking dyes, allowing PCR products to be directly loaded for electrophoresis.
5. High-Throughput Compatibility: Both lysis and amplification steps can be performed in a 96-well plate format, making it suitable for high-throughput screening.
Protocol
General Protocol
1. Genomic DNA release from animal tissue
1) Add 200 μL Lysis Buffer and 2 μL Proteases to a centrifuge tube and gently vortex to mix.
2) Add approximately 10 mg animal tissue (e.g., about 2 mm mouse tail, or an appropriate amount of mouse ear/toe, skin, internal organ, insect leg/wing, etc.) and gently vortex.
3) Incubate at 55°C for 5-30 min, followed by 95°C for 5 min.
4) Centrifuge at 12,000 rpm for 2 min.
5) Transfer the supernatant to a new tube for direct PCR amplification or storage at -20°C.
Note: a. Use the Lysis Buffer/Proteases mixture promptly. For multiple samples, prepare at a ratio of 100 μL Lysis Buffer : 1 μL Proteases;
b. Use a small amount of tissue and mince when possible. Recommended examples include 2-4 mm mouse tail, 1-4 toes, and approximately 2-4 mm pieces of mouse organ or brain. For small samples such as zebrafish, nematodes, or fruit flies, Lysis Buffer may be reduced to 50-100 μL; additional Proteases may be used for hard-shelled insect samples;
c. A 5 min incubation at 55°C is sufficient for most PCR applications. Extend to 30 min or longer for difficult tissues or higher DNA yield. Complete dissolution of tissue is not required.
2. PCR reaction system
| Component | Volume (20 μL) | Final concentration |
|---|---|---|
| Direct PCR Master Mix (2×, with Dye) | 10 μL | 1× |
| Forward Primer (10 μM) | 0.5 μL | 0.25 μM |
| Reverse Primer (10 μM) | 0.5 μL | 0.25 μM |
| Lysate (DNA template) | 2 μL | - |
| PCR Enhancer (5×) | 0-4 μL | 0-1× |
| ddH2O | To 20 μL | - |
Note: a. Use lysate at approximately 5%-20% of the total reaction volume and avoid exceeding 20%;
b. A final primer concentration of 0.2-0.25 μM is recommended and may be optimized within 0.1-0.5 μM;
c. PCR Enhancer may be added for difficult or complex templates. Reduce ddH2O accordingly.
3. PCR cycling program
| Step | Temperature | Time | Cycles |
|---|---|---|---|
| Initial denaturation | 94°C | 5 min | 1 |
| Denaturation | 94°C | 10 sec | 35-40 |
| Annealing | 50-65°C | 20 sec | 35-40 |
| Extension | 72°C | 30-60 sec/kb | 35-40 |
| Final extension | 72°C | 5 min | 1 |
The Direct PCR Master Mix contains loading dye, so PCR products may be loaded directly onto an agarose gel.
Storage
Lysis Buffer: 4°C, 1 year. If not be used for an extended period, it is recommended to aliquot it for storage.
Proteases, Direct PCR Master Mix (2×, with Dye), PCR Enhancer (5×): -20°C, 1 year. Avoid repeated freeze-thaw cycles.
Attention
1. To prevent cross-contamination between samples, sampling tools should be thoroughly cleaned after each use. It is recommended to immerse the cutting edge or any part in direct contact with the sample in 2% sodium hypochlorite solution, rinse repeatedly, and then wipe dry with clean tissue before reuse. For improved efficiency, multiple sets of sampling tools may be prepared and cleaned collectively to ensure that each sample is handled with contamination-free instruments.
2. Freshly collected animal tissues are recommended for optimal results. For long-term frozen samples, repeated freeze–thaw cycles should be avoided, as they may lead to genomic DNA degradation and consequently affect PCR amplification efficiency and data reliability.
3. This product is for R&D use only, not for drug, household, or other uses.
4. For your safety and health, please wear a lab coat and disposable gloves to operate.
Components
| Cat. No. | Product List | Components | HY-K0538-50 T | Storage |
|---|---|---|---|---|
| HY-K0538-A | Animal Tissue Direct PCR Reagent A | Lysis Buffer | 10 mL | 4°C, 1 year. |
| HY-K0538-B | Animal Tissue Direct PCR Reagent B | Proteases Direct PCR Master Mix (2×, with Dye) PCR Enhancer (5×) |
100 μL 500 μL 200 μL |
-20°C, 1 year. Avoid repeated freeze-thaw cycles. |
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