SampleIN™ Direct PCR Kit
Applications
- Direct PCR without template purification.
- Mouse genotyping and knockout analysis.
- Direct PCR from blood, mouse tail or ear, mammalian tissues (including FFPE).
- Direct PCR from hair follicle, buccal swabs, plant samples.
Benefits
- Ready-to load PCR in 50 minutes without template purification.
- Single-tube 15 min DNA extraction combined with fast hot-start PCR.
- Red dye in the PCR master mix for direct gel loading.
- High yields under standard or fast cycling conditions.
- Success with GC/AT rich templates.
SampleIN™ Direct PCR Kit
Product information "SampleIN™ Direct PCR Kit"
SampleIN™ Direct PCR Kit includes two modules: Two Lysis/Protease Buffers (Lysis Set for PCR/qPCR) and Hot Start PCR Master Mix.
SampleIN™ Direct PCR Kit is a premium tool for a fast direct PCR eliminating the need of tedious template purification. The kit is excellent for direct PCR from mouse tail or ear, mammalian tissues, hair follicle, buccal swabs and blood.
More information:
How to perform direct or crude sample PCR, qPCR and RT-qPCR
Using clinical or environmental samples without nucleic acids extraction.
Why direct PCR is not always working?
Quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) are fast techniques enabling detection and quantification of pathogens and nucleic acids. Direct amplification from crude samples avoids tedious extraction, but crude samples often contain natural PCR inhibitors that reduce efficiency, yield and reproducibility.
Short sample lysis combined with a swift protease digestion (for example using highQu SampleIN™ Lysis Set for PCR/qPCR) generates PCR/qPCR-ready templates. Crude extracts can be refrigerated and used for days to weeks.
Common PCR inhibitors in environmental & clinical crude samples
| Sample Type | Common Inhibitors |
|---|---|
| Blood | Hemoglobin, lactoferrin, immunoglobulins; heparin (anticoagulant) |
| Urine | Urea, salts, organic acids |
| Buccal swabs / Saliva | Mucins, proteases, food residues (polysaccharides, fats) |
| Plants | Polyphenols, polysaccharides (pectin), secondary metabolites, humic acids, seed lipids |
| Soil | Humic/fulvic acids, metal ions, polysaccharides, polyphenols |
Inhibition minimization & direct PCR optimization (brief)
- Use robust, inhibitor-resistant PCR/qPCR mixes that contain inhibitor-reducing and PCR-enhancing agents.
- If direct PCR fails without lysis, perform dilution series (e.g. 1:10 and 1:100). Rule of thumb: less is better than more.
- Always run positive controls with different dilutions of crude extract to determine at which concentration inhibitors appear.
- If direct PCR fails, perform lysis per kit manual and repeat dilution series. Optimize lysis and PCR parameters.
General rapid lysis procedure (highQu SampleIN™ Lysis Set for PCR/qPCR)
1. Sample collection & guidelines
| Sample (fresh or frozen) | Amount | Extraction vol. |
|---|---|---|
| Mouse tail | 2 mm or 3–5 mg | 100 µl |
| Mouse ear | 2 mm² or 3–5 mg | 100 µl |
| Mammalian tissue | 5 mg | 100 µl |
| FFPE tissue | 2 mm² of a 10 µm section | 100 µl |
| Blood (fresh / EDTA) | 2 µl | 100 µl |
| Blood Guthrie / FTA cards | 2 mm² | 100 µl |
| Hair follicle | 2 follicles | 100 µl |
| Buccal swab | 1 swab | 300 µl |
| Plant leaf | 2 mm², well crushed | 100 µl |
| Plant seed | Very small seed or 2 mm² piece, crushed | 100 µl |
Note: Increasing sample amounts slightly can improve yield but too much material may cause inefficient lysis and inhibition. Plant materials often require thorough mechanical disruption; optimization recommended.
2. Sample DNA lysis protocol (example)
- Use contamination-prevention measures: clean bench, gloves, sterile tubes.
- Thaw DPK buffers at room temperature and mix well.
- Prepare a 100 µl extraction reaction (use 3× volumes for buccal swabs):
DPK Lysis Buffer, 5× 20 µl DPK Protease Buffer, 10× 10 µl PCR water (not supplied) 70 µl - Mix gently. Place in thermal block / water bath:
Lysis: 75 °C — 5 min (vortex twice during lysis).
Protease inactivation: 95 °C — 10 min. - Add 900 µl PCR water. Centrifuge 1 min to pellet debris. Transfer supernatant to a sterile tube and keep on ice.
- Store at −20 °C for months or use immediately. Best results if PCR/qPCR performed immediately. Use supernatant directly as template (dilute 1:10 or more).
3. qPCR reaction setup (example)
Typical qPCR (20 µL total):
| Component | Final concentration / Notes |
|---|---|
| 4× qPCR master mix | 1× — use inhibitor-tolerant formulations |
| Forward primer | 200–400 nM (optimize) |
| Reverse primer | 200–400 nM |
| Probe (if used) | 100–250 nM |
| Template (crude lysate) | Typically 1–2 µl (test serial dilutions 1:10, 1:100) |
Controls setup
- No Template Control (NTC): detects contamination — replace template with water.
- No Reverse Transcriptase Control (−RT): for RNA templates — detects genomic DNA.
- Positive control: known pure template (purified or synthetic) to validate efficiency.
- Dilution control: test 1:10 and 1:100 template dilutions in duplicates/triplicates to reveal inhibitory effects.
- Internal Amplification Control (IAC): add a non-competitive synthetic target to detect inhibition and determine the concentration where inhibition occurs.
Template dilution advice
Start testing dilutions: 1:1, 1:10 and 1:100. Choose the lowest dilution that still gives robust amplification with acceptable Cq values. In RT-qPCR, avoid over-dilution that reduces sensitivity.
How to maintain PCR yield & reproducibility
- Keep the same optimized reaction setup and reagents (even same water) once established.
- Run reactions in duplicates or triplicates to reduce pipetting variance.
- Mix reagents well before use and maintain consistent sample input across replicates.
- Standardize lysis & dilution procedures. Avoid repeated freeze-thaw cycles — store crude extracts at +4 °C for short-term use.
Direct PCR/qPCR troubleshooting (quick guide)
| Issue | Possible cause | Solution |
|---|---|---|
| No amplification | Strong inhibition or suboptimal cycling | Increase dilution, re-lyse using less material; run annealing temperature gradient. |
| High Cq / weak signal | Low template or partial inhibition | Increase template input or prolong lysis to enrich targets. |
| Variable replicates | Pipetting error or non-homogeneous sample | Mix thoroughly, use master mixes, check plate/seal quality. |
| Amplification in NTC | Contamination | Replace reagents, clean workspace, use filtered tips. |
| Low efficiency (<85%) | Inhibitors or poor primer design | Optimize primers, increase dilution, or re-lyse with less sample material. |
| RNA degradation (RT-qPCR) | RNases in sample | Add RNase inhibitor before lysis, keep samples cold, shorten handling time, perform PCR immediately. |
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- Download Protocol and Specifications - Product Insert SampleIN™ Direct PCR Kit
- Protocol for Plant Samples
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- Download Protocol and Specifications - Product Insert SampleIN™ Direct PCR Kit
- Download MSDS SampleIN™ Direct PCR Kit
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- Have more specific questions? Contact us
- Download Protocol and Specifications - Product Insert SampleIN™ Direct PCR Kit
- Download MSDS SampleIN™ Direct PCR Kit
- Download highQu Catalogue of Premium Research Tools
- Download a list of Publications mentioning highQu products
- Or see how others use our products at bioz.com or google scholar
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- Have questions? Contact us
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