DirectPCR® Lysis Reagent (Tail)
A convenient lysis reagent for direct PCR preparation from mouse tail samples, designed to simplify genomic DNA preparation without conventional DNA purification.
DirectPCR-Tail
DirectPCR Lysis Reagent for mouse tail samples. Supplied as 100 ml, corresponding to the listed processing capacity of approximately 500 mouse tails.
DirectPCR® Lysis Reagent for Tail Samples
Simplify genomic DNA preparation for PCR from mouse tail tissue with a direct lysis workflow.
Product Overview
DirectPCR® Lysis Reagent (Tail) is designed for direct PCR sample preparation from mouse tail tissue. The workflow uses enzymatic tissue digestion followed by heat treatment to generate crude lysates suitable for PCR amplification.
The approach avoids the need for a conventional DNA purification procedure before PCR. After lysis and proteinase K inactivation, a small volume of crude lysate can be introduced into a PCR reaction.
Complete tissue lysis and appropriate proteinase K inactivation are important for reliable PCR performance.
Recommended Use
The reagent is intended for laboratory workflows in which genomic DNA from mouse tail tissue is required for PCR-based analysis.
A typical starting condition uses approximately 0.5 cm of tail tissue with 200–300 µl of DirectPCR Lysis Reagent containing freshly prepared Proteinase K.
For best performance, sample volume, tissue size and lysis conditions should be optimized for the specific laboratory workflow.
Key Features
Designed to provide a streamlined route from tissue sample to PCR-ready crude lysate.
Direct PCR Workflow
Crude lysates can be used directly in PCR after appropriate lysis and proteinase K inactivation.
Mouse Tail Samples
Formulated for workflows involving mouse tail tissue, with approximately 0.5 cm suggested as a starting sample size.
Simple Sample Preparation
The workflow combines tissue digestion and heat treatment without requiring conventional DNA purification before PCR.
Scalable Reagent Volume
Reagent volume can be adjusted approximately in proportion to the size of the tissue sample.
Crude Lysate Storage
According to the technical information, crude lysates may be stored at −20°C for up to one year or at 4°C for up to one week.
DNA Recovery Option
DNA from crude lysates can also be recovered by salt and isopropanol precipitation when further downstream analysis is required.
DirectPCR® Tail Lysis Workflow
Suggested starting conditions for preparation of mouse tail lysates for PCR.
Important: Proteinase K is required but is not included with the DirectPCR Lysis Reagent.
Add DirectPCR Lysis Reagent and Proteinase K
For a 0.5 cm tail, add 200–300 µl DirectPCR Lysis Reagent (Tail) containing freshly prepared 0.2–0.4 mg/ml Proteinase K.
Proteinase K is not included. When weighing Proteinase K powder is impractical for small numbers of samples, genomic PCR-quality Proteinase K solution can be used at 0.5–1.0 mg/ml.
Digest the Tissue
Rotate tubes in a rotating hybridization oven at 55°C for approximately 5–6 hours or until no visible tissue clumps remain.
Rotation may be continued overnight when necessary. Complete lysis is important for PCR performance.
Inactivate Proteinase K
Incubate the crude lysates at approximately 85°C for 45 minutes using a water bath or equivalent heating setup.
Proteinase K inactivation is an important step before adding crude lysate to PCR because residual proteinase K can interfere with PCR enzymes.
Add Lysate to PCR
Use approximately 0.5–1.0 µl of crude lysate in a 50 µl PCR reaction as a starting condition.
The technical information recommends several commercially available Taq polymerases for PCR workflows using the reagent.
Suggested Starting Conditions
Reference conditions for mouse tail tissue.
| Tail Size | DirectPCR Reagent | Dilution | Lysate / 50 µl PCR |
|---|---|---|---|
| 0.2–0.3 cm | 150–200 µl | 2-fold | 1.0–2.0 µl |
| 0.4 cm | 150–250 µl | 1-fold | 0.5–1.0 µl |
| 0.5 cm | 200–300 µl | 1-fold | 0.5–1.0 µl |
DNA Recovery from Crude Lysates
DNA can be recovered from DirectPCR crude lysates when purified DNA is required for additional analysis.
Optional DNA Rescue Procedure
Add NaCl to a final concentration of 250 mM, followed by 0.7 volume of isopropanol. DNA will form a precipitate. Centrifuge at 4°C for approximately 2 minutes, discard the supernatant and wash the DNA with 1 ml of 70% ethanol. Dissolve the recovered DNA in 50 µl of 10 mM Tris-HCl, pH 8.0. Approximately 1 µl can then be used for PCR.
Important Technical Tips
Practical considerations for consistent DirectPCR sample preparation.
Large tissue clumps should not remain after digestion. Vigorous shaking for several seconds during digestion can help disperse partially digested tissue and improve contact with the lysis reagent.
Heat inactivation at approximately 85–86°C for 45–50 minutes is described as critical for protecting Taq polymerase from residual Proteinase K activity.
The technical information identifies several commercially available Taq polymerases that were tested and recommended for this workflow.
A tail size of approximately 0.5 cm or slightly smaller is recommended as a starting point. Use a minimal crude lysate volume for PCR.
Smaller tubes can help minimize evaporation. The technical information recommends 0.75 ml tubes for lower reagent volumes.
When less than 50 µl of reagent is required, the reagent can be diluted with water by up to two-fold while maintaining the same Proteinase K concentration.
A rotating hybridization oven is described as performing better than a rocking plate for this tissue-lysis workflow.
Too much DirectPCR reagent or crude lysate can reduce PCR efficiency. Start with a small lysate volume and optimize when necessary.
Related DirectPCR® Products
Additional DirectPCR reagents for different biological sample types.
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