DirectPCR Lysis Reagent (Tail) for One-Step PCR Genotyping
Introduction
Extracting DNA from animal tissues can be challenging because certain compounds naturally inhibit PCR reactions. DirectPCR Lysis Reagents (Patent Pending) are specifically formulated to neutralize these PCR inhibitors, allowing DNA released from tissues to be directly compatible with one-step PCR genotyping. This eliminates time-consuming DNA purification steps and simplifies workflows in molecular biology, genetics, and transgenic research.
Product Overview
Product Name: DirectPCR Lysis Reagent (Tail)
Catalog Numbers: 101-T, 102-T
Application: Mouse tail genotyping
Volume per Use: 200–300 µL per 0.5 cm tail
The reagent works in combination with Proteinase K, which digests tissue proteins and enhances DNA release. The resulting lysates are stable, PCR-ready, and can also be stored long-term for later analyses.
Workflow: Mouse Tail DNA Extraction & PCR
1. Sample Preparation
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Use 0.5 cm mouse tail per tube.
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Add 200–300 µL DirectPCR Lysis Reagent (Tail) containing freshly prepared 0.2–0.4 mg/mL Proteinase K.
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For small numbers of samples, Proteinase K solution (Viagen, 0.5–1 mg/mL) can be used directly.
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Higher volumes (250–300 µL) improve lysis efficiency. Use 0.75 mL tubes for better mixing.
2. Tissue Digestion
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Incubate in a rotating hybridization oven at 55°C for 5–6 hours (or overnight if necessary).
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Ensure complete lysis: re-position or shake tubes after 2–3 hours to avoid tissue clumps.
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Note: Rotating oven > rocking plate for consistent results. DNA fragmentation from prolonged rotation does not impair PCR.
3. Proteinase K Inactivation
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Incubate lysates at 85°C for 45 min in a water bath.
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Optional: centrifuge briefly to remove hair or debris.
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Lysates can be stored at -20°C (up to 1 year) or 4°C (up to 1 week) without losing PCR efficacy.
4. PCR Amplification
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Use 0.5–1 µL of lysate per 50 µL PCR reaction.
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Recommended polymerases:
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Eppendorf HotMaster Taq
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Sigma JumpStart Taq
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Qiagen HotStar Taq
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Tip: Avoid using excessive lysate; too much DirectPCR reagent can inhibit PCR.
5. Optional DNA Rescue
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Precipitate DNA by adding NaCl to 250 mM and 0.7 volume of isopropanol.
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Centrifuge at 4°C, wash with 70% ethanol, and resuspend in 10 mM Tris-HCl (pH 8.0).
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Use 1 µL for PCR.
Suggested Starting Conditions
| Tail Size (cm) | DirectPCR (µL) | Dilution (fold) | Lysate per 50 µL PCR (µL) |
|---|---|---|---|
| 0.2–0.3 | 150–200 | 2 | 1.0–2.0 |
| 0.4 | 150–250 | 1 | 0.5–1.0 |
| 0.5 | 200–300 | 1 | 0.5–1.0 |
Related Products
| Product | Description | Cat # | Price (US$) |
|---|---|---|---|
| DirectPCR-Tail | 500 mouse tails (100 mL) | 102-T | 139 |
| DirectPCR-Ear | 500 mouse ears (50 mL) | 402-E | 139 |
| DirectPCR-Yolk sac | Yolk sac (100 mL) | 202-Y | 139 |
| DirectPCR-Cell | Cultured cells (100 mL) | 302-C | 139 |
| Proteinase K Solution | All tissue types (100 mg) | 501-PK | 99 |
Technical Tips for Optimal Results
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Ensure complete lysis; tissue clumps reduce DNA yield.
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Proper Proteinase K inactivation protects Taq polymerase.
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Use recommended Taq polymerases for consistent amplification.
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Keep tail size ≤0.5 cm and lysate volumes minimal for PCR efficiency.
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Use small tubes (0.75 mL) to minimize evaporation for low-volume samples.
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Dilute DirectPCR reagent if volumes are very small, adjusting lysate proportionally.
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PCR machine reliability is critical; faulty thermocyclers can compromise results.