One-Step Mouse Tissue Direct Amplification Kit contains both crude DNA extraction and PCR amplification systems, enabling direct and rapid PCR amplification of mouse tissue samples (such as tail, ear, and toe) for genotyping. It eliminates the need for overnight digestion, phenol-chloroform extraction, or column-based purification, greatly shortening experimental time. This product is suitable for amplification of target fragments up to 2 kb and multiplex PCR reactions with up to 3 primer pairs.
The 2× Mouse Tissue Direct PCR Mix component contains genetically engineered DNA polymerase, Mg²⁺, dNTPs, and an optimized buffer system, providing high amplification efficiency and excellent tolerance to inhibitors. Simply add template, primers, and water to perform PCR amplification with easy operation.
This product is suitable for mouse gene knockout analysis, transgenic identification, genotyping, etc. The amplification products have 3'-end A-overhangs (sticky ends) and can be directly used for TA cloning.
Contents & Storage
| Storage and Transport Unit Item No. | Storage and Transport Unit Name | Component No. | Component Name | Appearance | 500μL | 5×1mL | Storage | | O1527919DT | One Step Mouse Tissue Direct Amplification Kit (-20℃ Components) | O1527919A | 2×Mouse Tissue Direct PCR Mix | Liquid | 500μL | 5×1mL | -20℃. | | | O1527919B | Proteinase K | Liquid | 80μL | 800μL | -20℃. | | O1527919WT | One Step Mouse Tissue Direct Amplification Kit (2-8℃ Components) | O1527919C | Lysis Buffer | Liquid | 4mL | 2×20mL | 2-8℃. |
Note: Each component is individually packaged according to its storage temperature (2‑8 °C/-20 °C) upon shipment, with differentiated temperature‑controlled transportation to guarantee product performance. Orders containing materials requiring multiple temperature zones may be delivered in separate batches; please refer to the actual received goods. Upon receipt, store components separately at the temperatures specified in the component list. The shelf‑life is 24 months from the date of manufacture.
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Instructions
(I) Release of Genomic DNA from Samples
1.Prepare an appropriate amount of lysis buffer (1×) according to the number of samples to be lysed. The preparation method for a single sample is as follows:
| Component | Volume（μL） | | Lysis Buffer | 200 | | Proteinase K | 4 |
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2.Take an appropriate amount of mouse tissue sample (refer to the table below for tissue usage) into a clean centrifuge tube. Add 200 μL of freshly prepared tissue lysis buffer to each tube, vortex mix, and incubate at 55°C for 30 min, followed by heating at 98°C for 3 min (to inactivate Proteinase K). Recommended tissue amounts: mouse tail tip 1–3 mm, mouse ear 2–5 mm², and mouse toe 1–2 pieces.
3.Vortex the lysate thoroughly to mix well, centrifuge at 12,000 rpm (~13,400×g) for 5 min, and use the supernatant for PCR reaction. Alternatively, transfer the supernatant to a sterile centrifuge tube and store at –20°C for up to 2 weeks.
(II) PCR Amplification
1.Reaction setup: Remove 2× Mouse Tissue Direct PCR Mix from –20°C, thaw completely on ice, and mix by inverting. Prepare the reaction mixture according to the table below (perform on ice):
| Component | 25 μL System | 50 μL System | | 2× Mouse Tissue Direct PCR Mix | 12.5 μL | 25 μL | | Forward Primer（10 μM） | 1 μL | 2 μL | | Reverse Primer（10 μM） | 1 μL | 2 μL | | Lysate（Template DNA） | 1~3 μL | 2~5 μL | | ddH₂O | to 25 μL | to 50 μL |
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Note: The volume of lysate added should not exceed 1/10 of the total reaction system; excessive amount may inhibit PCR amplification.
2.Reaction program:
| Step | Temperature | Time | Cycles | | Initial Denaturation | 94℃ | 5 min | 1 | | Denaturation | 94℃ | 30 sec | 35~40 | | Annealing | Tm+3~5℃ | 30 sec | 35~40 | | Extension | 72℃ | 30 sec/kb | 35~40 | | Final Extension | 72℃ | 5 min | 1 | | Hold | 4℃ | Hold | — |
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Note: The annealing temperature should be set according to the Tm values of the primers. It is recommended to set the annealing temperature to the lower Tm value of the primers +3 to 5°C, with a maximum of ≤68°C.
Precautions
1.To avoid cross-contamination between samples, prepare multiple sampling tools. If tools are to be reused, clean their surfaces with 2% sodium hypochlorite solution or nucleic acid cleaning solution after each use.
2.It is recommended to use freshly collected mouse tissue. The sample amount should not be too large, as excessive amounts may affect the amplification results.
3.The lysis buffer should be stored at 2–8°C. If stored at low temperatures (≤0°C), precipitation may occur. Be sure to completely dissolve the precipitate before use.
4.The 2× Mouse Tissue Direct PCR Mix should be protected from repeated freeze-thaw cycles. 5.For frequent short-term use, it may be stored at 2–8°C.
This product is for research use only and is not intended for clinical diagnosis or other applications.
6.For your safety and health, please wear a lab coat and disposable gloves during operation.
FAQ
| Problem | Possible Cause | Suggestion | | No amplification or weak product | Excessive lysate | Choose the most appropriate template amount, generally not exceeding 1/10 of the system |
| Inefficient DNA release | Try extending the incubation time of lysis buffer with sample |
| Incomplete Proteinase K inactivation | Inactivation step can be performed in boiling water bath | | Non-specific amplification | Poor PCR primer quality | Verify primer quality using genomic DNA amplification; optimize primer design |
| Poor primer specificity | Optimize primers to avoid non-specific bands and primer dimer amplification |
| Excessive template concentration | Reduce template amount or dilute template 10-fold before amplification |
| Annealing temperature too low | Try increasing annealing temperature and set up annealing temperature gradient |
| High temperature during reaction setup | Prepare PCR reaction system on ice |
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