The HighScriber™ Reverse Transcriptase Mix is a premium tool for the high efficiency reverse transcription of up to 12-15 kb long cDNA. Mix includes HighScriber™ Reverse Transcriptase and Ribonuclease Inhibitor for save, robust cDNA synthesis and ease of use. HighScriber™ Reverse Transcriptase allows for high detection sensitivity from 1 pg of total RNA. The wide reaction temperature range (38°C - 55°C) ensures efficient cDNA synthesis from complex or GC rich templates. The enzyme uses ssRNA or ssDNA as a template, possesses no detectable Ribonuclease H activity specific to RNA in RNA-DNA hybrids. A highly reduced Ribonuclease H activity allows for transcription of full lengths long transcripts. HighScriber™ Reverse Transcriptase can be used for RACE as it has terminal transferase activity - adds cytosines to 3’ ends of cDNA. The Ribonuclease inhibitor premixed with the RT ensures RNA protection from ribonuclease degradation. Supplied 5X ALLin™ HighScriber Buffer includes everything you need for the cDNA synthesis reaction. To minimize pipetting steps it contains MgCl2, dNTPs, enhancers, stabilizers. The only things to add is the template RNA and primer.

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.

SecurRIN™ Advanced RNase Inhibitor is a premium tool for RNA protection from degradation during enzymatic reactions, storage or extraction. It is an extraordinary stable and robust enzyme: it works at up to 60°C temperature, remains active after weeks of room temperature exposure and multiple freezing thawing cycles. It is active in different buffer conditions within a broad pH range of 5.5 to 9.0, and within 0.5 - 1 mM concentration of DTT. SecurRIN™ Advanced RNase Inhibitor is a 50 kDa non-covalent inhibitor of RNase A, RNase B, and RNase C binding them in a 1 to 1 ratio. It is a recombinant protein derived from E. coli strain carrying human placenta RNase Inhibitor gene. SecurRIN™ Advanced RNase Inhibitor does not inhibit RNAses H, 1, T1, T2 and S1 Nuclease. It influences neither the activity nor the performance of DNA polymerases and of Reverse Transcriptases. The enzyme is free from DNAses and RNAses. Ribonuclease Inhibitor is a protein that protects RNA samples from degradation by ribonucleases. Ribonucleases are present everywhere in laboratory environment, they are very robust and ubiquitous enzymes that rapidly degrade RNA by destroying valuable RNA samples during molecular biology or diagnostics workflows. RNase inhibitor binds to RNases and inactivates these enzymes, ensuring the integrity and stability of RNA samples during such processes as RNA isolation, clean-up, cDNA synthesis, RT-qPCR and other applications. A good RNase inhibitor should have high affinity for RNases and strong inhibitory activity under a wide variety of buffer conditions. It shall tolerate different pH values, salt concentrations and shall have high affinity to different types of RNases. It should also be stable under higher temperatures and easy to use in all common RNA-related buffers with minimal interference with downstream applications. Furthermore, it is especially important, that the Inhibitor itself is pure and free from contaminating RNases. highQu offers SecurRIN™ Advanced RNase Inhibitor for RNA protection from degradation during enzymatic reactions, storage or extraction. Is is a 50 kDa non-covalent inhibitor of RNase A, RNase B, and RNase C.  

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 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 reactionChemicals 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-qPCRUsing 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 samplesSample TypeCommon InhibitorsBloodHemoglobin, lactoferrin, immunoglobulins; heparin (anticoagulant)UrineUrea, salts, organic acidsBuccal swabs / SalivaMucins, proteases, food residues (polysaccharides, fats)PlantsPolyphenols, polysaccharides (pectin), secondary metabolites, humic acids, seed lipidsSoilHumic/fulvic acids, metal ions, polysaccharides, polyphenolsInhibition 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 & guidelinesSample (fresh or frozen)AmountExtraction vol.Mouse tail2 mm or 3–5 mg100 µlMouse ear2 mm² or 3–5 mg100 µlMammalian tissue5 mg100 µlFFPE tissue2 mm² of a 10 µm section100 µlBlood (fresh / EDTA)2 µl100 µlBlood Guthrie / FTA cards2 mm²100 µlHair follicle2 follicles100 µlBuccal swab1 swab300 µlPlant leaf2 mm², well crushed100 µlPlant seedVery small seed or 2 mm² piece, crushed100 µlNote: 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 µlDPK Protease Buffer, 10×10 µlPCR water (not supplied)70 µlMix 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):ComponentFinal concentration / Notes4× qPCR master mix1× — use inhibitor-tolerant formulationsForward primer200–400 nM (optimize)Reverse primer200–400 nMProbe (if used)100–250 nMTemplate (crude lysate)Typically 1–2 µl (test serial dilutions 1:10, 1:100)Controls setupNo 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 adviceStart 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 & reproducibilityKeep 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)IssuePossible causeSolutionNo amplificationStrong inhibition or suboptimal cyclingIncrease dilution, re-lyse using less material; run annealing temperature gradient.High Cq / weak signalLow template or partial inhibitionIncrease template input or prolong lysis to enrich targets.Variable replicatesPipetting error or non-homogeneous sampleMix thoroughly, use master mixes, check plate/seal quality.Amplification in NTCContaminationReplace reagents, clean workspace, use filtered tips.Low efficiency (<85%)Inhibitors or poor primer designOptimize primers, increase dilution, or re-lyse with less sample material.RNA degradation (RT-qPCR)RNases in sampleAdd 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 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 reactionChemicals 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-qPCRUsing 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 samplesSample TypeCommon InhibitorsBloodHemoglobin, lactoferrin, immunoglobulins; heparin (anticoagulant)UrineUrea, salts, organic acidsBuccal swabs / SalivaMucins, proteases, food residues (polysaccharides, fats)PlantsPolyphenols, polysaccharides (pectin), secondary metabolites, humic acids, seed lipidsSoilHumic/fulvic acids, metal ions, polysaccharides, polyphenolsInhibition 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 & guidelinesSample (fresh or frozen)AmountExtraction vol.Mouse tail2 mm or 3–5 mg100 µlMouse ear2 mm² or 3–5 mg100 µlMammalian tissue5 mg100 µlFFPE tissue2 mm² of a 10 µm section100 µlBlood (fresh / EDTA)2 µl100 µlBlood Guthrie / FTA cards2 mm²100 µlHair follicle2 follicles100 µlBuccal swab1 swab300 µlPlant leaf2 mm², well crushed100 µlPlant seedVery small seed or 2 mm² piece, crushed100 µlNote: 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 µlDPK Protease Buffer, 10×10 µlPCR water (not supplied)70 µlMix 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):ComponentFinal concentration / Notes4× qPCR master mix1× — use inhibitor-tolerant formulationsForward primer200–400 nM (optimize)Reverse primer200–400 nMProbe (if used)100–250 nMTemplate (crude lysate)Typically 1–2 µl (test serial dilutions 1:10, 1:100)Controls setupNo 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 adviceStart 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 & reproducibilityKeep 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)IssuePossible causeSolutionNo amplificationStrong inhibition or suboptimal cyclingIncrease dilution, re-lyse using less material; run annealing temperature gradient.High Cq / weak signalLow template or partial inhibitionIncrease template input or prolong lysis to enrich targets.Variable replicatesPipetting error or non-homogeneous sampleMix thoroughly, use master mixes, check plate/seal quality.Amplification in NTCContaminationReplace reagents, clean workspace, use filtered tips.Low efficiency (<85%)Inhibitors or poor primer designOptimize primers, increase dilution, or re-lyse with less sample material.RNA degradation (RT-qPCR)RNases in sampleAdd 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 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. 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 reactionChemicals 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-qPCRUsing 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 samplesSample TypeCommon InhibitorsBloodHemoglobin, lactoferrin, immunoglobulins; heparin (anticoagulant)UrineUrea, salts, organic acidsBuccal swabs / SalivaMucins, proteases, food residues (polysaccharides, fats)PlantsPolyphenols, polysaccharides (pectin), secondary metabolites, humic acids, seed lipidsSoilHumic/fulvic acids, metal ions, polysaccharides, polyphenolsInhibition 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 & guidelinesSample (fresh or frozen)AmountExtraction vol.Mouse tail2 mm or 3–5 mg100 µlMouse ear2 mm² or 3–5 mg100 µlMammalian tissue5 mg100 µlFFPE tissue2 mm² of a 10 µm section100 µlBlood (fresh / EDTA)2 µl100 µlBlood Guthrie / FTA cards2 mm²100 µlHair follicle2 follicles100 µlBuccal swab1 swab300 µlPlant leaf2 mm², well crushed100 µlPlant seedVery small seed or 2 mm² piece, crushed100 µlNote: 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 µlDPK Protease Buffer, 10×10 µlPCR water (not supplied)70 µlMix 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):ComponentFinal concentration / Notes4× qPCR master mix1× — use inhibitor-tolerant formulationsForward primer200–400 nM (optimize)Reverse primer200–400 nMProbe (if used)100–250 nMTemplate (crude lysate)Typically 1–2 µl (test serial dilutions 1:10, 1:100)Controls setupNo 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 adviceStart 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 & reproducibilityKeep 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)IssuePossible causeSolutionNo amplificationStrong inhibition or suboptimal cyclingIncrease dilution, re-lyse using less material; run annealing temperature gradient.High Cq / weak signalLow template or partial inhibitionIncrease template input or prolong lysis to enrich targets.Variable replicatesPipetting error or non-homogeneous sampleMix thoroughly, use master mixes, check plate/seal quality.Amplification in NTCContaminationReplace reagents, clean workspace, use filtered tips.Low efficiency (<85%)Inhibitors or poor primer designOptimize primers, increase dilution, or re-lyse with less sample material.RNA degradation (RT-qPCR)RNases in sampleAdd 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 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. 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 reactionChemicals 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-qPCRUsing 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 samplesSample TypeCommon InhibitorsBloodHemoglobin, lactoferrin, immunoglobulins; heparin (anticoagulant)UrineUrea, salts, organic acidsBuccal swabs / SalivaMucins, proteases, food residues (polysaccharides, fats)PlantsPolyphenols, polysaccharides (pectin), secondary metabolites, humic acids, seed lipidsSoilHumic/fulvic acids, metal ions, polysaccharides, polyphenolsInhibition 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 & guidelinesSample (fresh or frozen)AmountExtraction vol.Mouse tail2 mm or 3–5 mg100 µlMouse ear2 mm² or 3–5 mg100 µlMammalian tissue5 mg100 µlFFPE tissue2 mm² of a 10 µm section100 µlBlood (fresh / EDTA)2 µl100 µlBlood Guthrie / FTA cards2 mm²100 µlHair follicle2 follicles100 µlBuccal swab1 swab300 µlPlant leaf2 mm², well crushed100 µlPlant seedVery small seed or 2 mm² piece, crushed100 µlNote: 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 µlDPK Protease Buffer, 10×10 µlPCR water (not supplied)70 µlMix 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):ComponentFinal concentration / Notes4× qPCR master mix1× — use inhibitor-tolerant formulationsForward primer200–400 nM (optimize)Reverse primer200–400 nMProbe (if used)100–250 nMTemplate (crude lysate)Typically 1–2 µl (test serial dilutions 1:10, 1:100)Controls setupNo 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 adviceStart 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 & reproducibilityKeep 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)IssuePossible causeSolutionNo amplificationStrong inhibition or suboptimal cyclingIncrease dilution, re-lyse using less material; run annealing temperature gradient.High Cq / weak signalLow template or partial inhibitionIncrease template input or prolong lysis to enrich targets.Variable replicatesPipetting error or non-homogeneous sampleMix thoroughly, use master mixes, check plate/seal quality.Amplification in NTCContaminationReplace reagents, clean workspace, use filtered tips.Low efficiency (<85%)Inhibitors or poor primer designOptimize primers, increase dilution, or re-lyse with less sample material.RNA degradation (RT-qPCR)RNases in sampleAdd 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.