highQu ALLin™ Taq DNA Polymerase is the versatile engineered enzyme which in combination with the optimized ALLin™ buffer provides higher success rates in demanding PCR applications like amplification of complex templates, crude sample PCR and fast cycling. ALLin™ Taq DNA Polymerase has the same PCR accuracy like Taq DNA Polymerase, 4.5 x 10^4 (a number of correct nucleotides incorporated before the error occurs) and produces A-tailed products suitable for ligating into TA cloning vectors. The convenience of ALLin™ Taq DNA Polymerase is maximized by the use of 2X Red Mastermix providing the additional advantage of reduced pipetting and minimized errors. ALLin™ Red Taq Mastermix, 2X is premixed with red dye and density reagents for direct loading on the gels after the PCR. In a 2% agarose TAE gel the dye migrates with~350 bp DNA, in 1% agarose TAE gel with ~ 600 bp DNA fragments. The mastermix is even supplied with the PCR Water, and the only thing to add is the template with primers.

The SampleIN™ Direct qPCR Probe Mix has been specifically designed for use with unpurified samples or crude lysates as templates. This 4X concentrated qPCR master mix with the Hot Start Taq DNA Polymerase, dNTPs, magnesium, and optimized buffer delivers an exceptional PCR inhibitor tolerance in direct qPCR applications. It includes PCR enhancers and stabilizers and is formulated to provide the robust performance with such common sample materials like unpurified saliva or fresh blood. The mix tolerates a range of common chemicals present in purified DNA templates such as guanidine, alcohols, SDS and similar, as well as common blood, urine and environmental natural sample-compounds known to inhibit PCR such as hemoglobin, immunoglobulins, heparin, urea, polyphenols, cellulose, humic and tannic acids and chlorophyl. The Lyo-ready version is available for production of room temperature stable kits: Lyo-Ready SampleIN™ Direct qPCR Probe Mix. It has been specifically designed for use with unpurified sample specimens or crude lysates as templates. Inquire for OEM pricing now. The mix can be used with all probe types and with a variety of sample amounts from a very low-copy number targets in diluted samples to abundant templates.  This Direct qPCR Probe Mix contains all compounds required for robust qPCR reaction, and the only components to add are template, primers and probes. The high concentration of the master mix enables the use of maximal template volume as well as multiple probes and primers. Mix is supplied with PCR Water. Though the SampleIN™ Direct qPCR Probe Mix has been successfully tested for the use with fresh blood and urine samples; for more consistent results, it is always recommended to purify the template, or at least to perform fast lysis using highQu SampleIN™ Lysis Set for PCR/qPCR. For work with crude or inhibitor-rich RNA templates, we offer a SampleIN™ 1Step RT qPCR Probe Mixes, 4X with reverse transcriptase.  The SampleIN™ Direct qPCR Probe Mix does not include ROX, the version with Low ROX concentration is available as SampleIN™ Direct qPCR Probe ROX L Mix. If high ROX concentration or more ROX flexibility is required, ROX for qPCR Mixes, 50 μM can be obtained separately and added directly into the qPCR mix. Tolerates common inhibitors, such as: 5-7% crude saliva and crude blood in the reaction Chemicals left after NA extractions (guanidine, alcohols, SDS)  Blood compounds (hematin, hemoglobin, hemin, immunoglobulins) Saliva and urine compounds (urea) Plant, soil samples (chlorophyl, humic, tannic acids, quercetin cellulose)    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. This guide provides general recommendations. Optimize lysis and PCR parameters for your specific samples and targets. highQu inhibitor-resistant mixes tolerate traces of chlorophyll, polyphenols, tannic acid, cellulose and pectin, but plant samples often require stronger mechanical disruption or specific extraction methods. ```

SampleIN™ Direct qPCR Probe ROX L Mix has been specifically designed for use with unpurified samples or crude lysates as templates. This 4X concentrated qPCR master mix with the Hot Start Taq DNA Polymerase, dNTPs, magnesium, and optimized buffer delivers an exceptional PCR inhibitor tolerance in direct qPCR applications. It includes PCR enhancers and stabilizers and is formulated to provide the robust performance with such common sample materials like unpurified saliva or fresh blood. The mix tolerates a range of common chemicals present in purified DNA templates such as guanidine, alcohols, SDS and similar, as well as common blood, urine and environmental natural sample-compounds known to inhibit PCR such as hemoglobin, immunoglobulins, heparin, urea, polyphenols, cellulose, humic and tannic acids and chlorophyl. The Lyo-ready version is available for production of room temperature stable kits: Lyo-Ready SampleIN™ Direct qPCR Probe Mix. It has been specifically designed for use with unpurified sample specimens or crude lysates as templates. Inquire for OEM pricing now. The mix can be used with all probe types and with a variety of sample amounts from a very low-copy number targets in diluted samples to abundant templates.  This Direct qPCR Probe Mix contains all compounds required for robust qPCR reaction, and the only components to add are template, primers and probes. The high concentration of the master mix enables the use of maximal template volume as well as multiple probes and primers. Mix is supplied with PCR Water. Though the SampleIN™ Direct qPCR Probe Mix has been successfully tested for the use with fresh blood and urine samples; for more consistent results, it is always recommended to purify the template, or at least to perform fast lysis using highQu SampleIN™ Lysis Set for PCR/qPCR. For work with crude or inhibitor-rich RNA templates, we offer a SampleIN™ 1Step RT qPCR Probe Mixes, 4X with reverse transcriptase.  SampleIN™ Direct qPCR Probe ROX L Mix includes ROX at low concentration, the version without ROX is available as SampleIN™ Direct qPCR Probe Mix. If high ROX concentration or more ROX flexibility is required, ROX for qPCR Mixes, 50 μM can be obtained separately and added directly into the qPCR mix. Tolerates common inhibitors, such as: 5-7% crude saliva and crude blood in the reaction Chemicals left after NA extractions (guanidine, alcohols, SDS)  Blood compounds (hematin, hemoglobin, hemin, immunoglobulins) Saliva and urine compounds (urea) Plant, soil samples (chlorophyl, humic, tannic acids, quercetin cellulose)  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. This guide provides general recommendations. Optimize lysis and PCR parameters for your specific samples and targets. highQu inhibitor-resistant mixes tolerate traces of chlorophyll, polyphenols, tannic acid, cellulose and pectin, but plant samples often require stronger mechanical disruption or specific extraction methods. ```  

SampleIN™ 1Step RT qPCR Probe Mix has been specifically designed for use with crude lysates and impure templates. This 4X concentrated single-tube RT qPCR master mix with the Hot Start Taq DNA Polymerase, Reverse Transcriptase, RNase inhibitor, dNTPs, magnesium and optimized buffer delivers an exceptional PCR inhibitor tolerance in direct one-step qPCR applications. It includes PCR enhancers and stabilizers and is formulated to provide the robust performance with such common sample materials like unpurified saliva or fresh blood. The mix tolerates a range of common chemicals present in purified DNA templates such as guanidine, alcohols, SDS and similar, as well as common blood, urine and environmental natural sample-compounds known to inhibit PCR such as hemoglobin, immunoglobulins, heparin, urea, polyphenols, cellulose, humic and tannic acids and chlorophyl. The Lyo-ready version is available for production of room temperature stable kits: Lyo-Ready SampleIN™ 1Step RT qPCR Probe Kit It has been specifically designed for use with unpurified sample specimens or crude lysates as templates. Inquire for OEM pricing now. Additionally, the air-dryabale version is available for production of room temperature stable kits: AirDry SampleIN™ 1Step RT qPCR Probe Mix, 4X. It has been specifically designed for use with unpurified sample specimens or crude lysates as templates. You can dry the assays without special lyophilization equipment. Inquire for OEM pricing now. This 1Step RT qPCR Probe Mix contains all compounds required for robust single-tube RT qPCR reaction, and the only components to add are template, primers and probes. The high concentration of the master mix enables the use of maximal template volume as well as multiple probes and primers. The Mix is supplied with PCR Water. SampleIN™ 1Step RT qPCR Probe Mix has been successfully tested for the use with fresh blood and urine samples; for more consistent results, it is always recommended to purify the template, or at least to perform fast lysis using highQu SampleIN™ Lysis Set for PCR/qPCR. For work with crude or inhibitor-rich DNA templates, we offer a SampleIN™ Direct qPCR Probe Mixes. SampleIN™ 1Step RT qPCR Probe Mix does not include ROX, the version with Low ROX concentration is available as SampleIN™ 1Step RT qPCR Probe ROX L Mix. If high ROX concentration or more ROX flexibility is required, ROX for qPCR Mixes, 50 μM can be obtained separately and added directly into the qPCR mix. Tolerates common inhibitors, such as: 5-7% crude saliva and crude blood in the reaction Chemicals left after NA extractions (guanidine, alcohols, SDS)  Blood compounds (hematin, hemoglobin, hemin, immunoglobulins) Saliva and urine compounds (urea) Plant, soil samples (chlorophyl, humic, tannic acids, quercetin cellulose)  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. This guide provides general recommendations. Optimize lysis and PCR parameters for your specific samples and targets. highQu inhibitor-resistant mixes tolerate traces of chlorophyll, polyphenols, tannic acid, cellulose and pectin, but plant samples often require stronger mechanical disruption or specific extraction methods. ```  

SampleIN™ 1Step RT qPCR Probe ROX L Mix has been specifically designed for use with crude lysates and impure templates. This 4X concentrated single-tube RT qPCR master mix with the Hot Start Taq DNA Polymerase, Reverse Transcriptase, RNase inhibitor, dNTPs, magnesium and optimized buffer delivers an exceptional PCR inhibitor tolerance in direct one-step qPCR applications. It includes PCR enhancers and stabilizers and is formulated to provide the robust performance with such common sample materials like unpurified saliva or fresh blood. The mix tolerates a range of common chemicals present in purified DNA templates such as guanidine, alcohols, SDS and similar, as well as common blood, urine and environmental natural sample-compounds known to inhibit PCR such as hemoglobin, immunoglobulins, heparin, urea, polyphenols, cellulose, humic and tannic acids and chlorophyl. The mix can be used with all probe types and with a variety of sample amounts from a very low-copy number targets in diluted samples to abundant templates. The Lyo-ready version is available for production of room temperature stable kits: Lyo-Ready SampleIN™ 1Step RT qPCR Probe Kit It has been specifically designed for use with unpurified sample specimens or crude lysates as templates. Inquire for OEM pricing now. Additionally, the air-dryabale version is available for production of room temperature stable kits: AirDry SampleIN™ 1Step RT qPCR Probe Mix, 4X. It has been specifically designed for use with unpurified sample specimens or crude lysates as templates. You can dry the assays without special lyophilization equipment. Inquire for OEM pricing now. This 1Step RT qPCR Probe Mix contains all compounds required for robust single-tube RT qPCR reaction, and the only components to add are template, primers and probes. The high concentration of the master mix enables the use of maximal template volume as well as multiple probes and primers. The Mix is supplied with PCR Water. SampleIN™ 1Step RT qPCR Probe Mix has been successfully tested for the use with fresh blood and urine samples; for more consistent results, it is always recommended to purify the template, or at least to perform fast lysis using highQu SampleIN™ Lysis Set for PCR/qPCR. For work with crude or inhibitor-rich DNA templates, we offer a SampleIN™ Direct qPCR Probe Mixes. SampleIN™ 1Step RT qPCR Probe ROX L Mix  includes ROX at low concentration, the version without ROX is available as SampleIN™ 1Step RT qPCR Probe Mix. If high ROX concentration or more ROX flexibility is required, ROX for qPCR Mixes, 50 μM can be obtained separately and added directly into the qPCR mix. Tolerates common inhibitors, such as: 5-7% crude saliva and crude blood in the reaction Chemicals left after NA extractions (guanidine, alcohols, SDS)  Blood compounds (hematin, hemoglobin, hemin, immunoglobulins) Saliva and urine compounds (urea) Plant, soil samples (chlorophyl, humic, tannic acids, quercetin cellulose)  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. This guide provides general recommendations. Optimize lysis and PCR parameters for your specific samples and targets. highQu inhibitor-resistant mixes tolerate traces of chlorophyll, polyphenols, tannic acid, cellulose and pectin, but plant samples often require stronger mechanical disruption or specific extraction methods. ```  

highQu qPCR master mixes are based on the small molecular inhibitor technology Hot Start PCR allowing to achieve highest sensitivity and specificity under both standard and fast qPCR cycling conditions. They provide excellent results on both AT and GC rich templates, in multiplexing and guaranty rapid extension with early Ct values with minimum or no optimization. Our master mixes are supplied with PCR Water to guaranty the best performance. To suit the broad instrument range the ORA™ qPCR Probe Master mixes are available in three versions – without ROX, with low or high ROX concentration.

highQu ALLin™ RPH Polymerase (Robust, Proofreading, Hot-start Polymerase) is the versatile engineered enzyme combining best polymerase properties for excellence in most demanding PCR applications, like low copy detection, long or high fidelity PCR, amplification of complex templates, crude sample PCR and multiplexing. ALLin™ RPH Polymerase has 5 times higher fidelity than Taq DNA Polymerase and produces A-tailed products suitable for ligating into TA cloning vectors. For the maximum convenience the ALLin™ RPH Mastermix, 2X is available.

Our improved 2X 1Step RT qPCR Probe Kits provide highly sensitive target RNA detection when working with a wide variety of samples. The optimized 1Step RT qPCR Pro Mix in combination with a blend of thermostable Reverse Transcriptase and an advanced RNase Inhibitor (RT-Pro Mix) allows for a single step, one tube RT qPCR with great results even in multiplex reactions. The novel RT-Pro Mix ensures safe and efficient RNA template conversion into a single-stranded cDNA. Pure RNA samples, as well as lysed crude samples can serve as templates for one-step RT qPCR. Though the kit was not specifically designed for crude sample qPCR, it has a potential to work well for this application. The robust 1Step RT qPCR Pro Mix is a 2X concentrated qPCR master mix which includes dNTPs, buffer, and the hot start Taq DNA Polymerase. The Hot Start function ensures the highest sensitivity and specificity under both standard and fast qPCR cycling conditions. The kits provide excellent results on both AT and GC-rich templates and show early Ct values with minimum or no optimization. Our kits include PCR Water to guaranty the best performance. To suit the broad instrument range the 2X 1Step RT qPCR Probe Kits are available in three versions – without ROX, with low or high ROX concentration.