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.

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.

Derived from our HiFi polymerase, the highQu ALLin™ Mega HiFi DNA Polymerase provides much lower error rate PCR with a 100 higher fidelity compared to Taq. The ALLin™ Mega HiFi DNA Polymerase is engineered to be much faster and to generate a higher yield of long PCR products up to 20 kb from complex GC-rich templates. Therefore the ALLin™ Mega HiFi DNA Polymerase is an excellent choice for longer and very complex PCR applications where the highest fidelity is demanded. It is an enzyme of choice for cloning and all kind of sequencing applications including NGS. Generated blunt-ended PCR products are suitable for ligation into blunt vectors. For maximum convenience, the ALLin™ Mega HiFi Mastermix, 2X (HLM0201) and ALLin™ Mega HiFi Red Mastermix, 2X (HLM0301) are available. Pfu DNA Polymerase was originally found in thermophilic bacterium Pyrococcus furiosus. It is a thermostable DNA polymerase that was widely used in molecular biology applications for its high fidelity of DNA amplification due to a high proofreading activity. Pfu polymerase has 3' to 5' exonuclease proofreading activity, which enables it to correct errors that occur during DNA synthesis. The enzyme is commonly used for cloning and sequencing applications, however it is relatively slow, sensitive to contaminants and inhibitors and has low processivity resulting in usually low PCR yields. Alternative, mostly better DNA polymerases have been developed by different companies to address the limitations of Pfu polymerase. These include such enzymes as Phusion® DNA polymerase, Q5® DNA polymerase from NEB, and many others. These enzymes offer improved elongation rates, reduced error rates, and increased efficiency for NGS applications. The ALLin™ MEGA HiFi Polymerase offered by highQu provides 100X higher fidelity compared to Taq Polymerase, which is much higher than Pfu. In addition, the hot start enzyme version allows to reduce the background to the minimum. Such robust enzyme offers not only precise amplification, but also enables PCR from very complex templates and generation of very long PCR products up to 30 kb and above. Availability of faster, more robust and more precise polymerases influenced successful development of next generation sequencing techniques (NGS) where PCR precision and yield is crucial for reliable sequencing results.

The ALLin™ Bst DNA Polymerase is a recombinant protein, representing a large fragment of the B. stearothermophilus DNA Polymerase expressed in E. coli cells. This robust polymerase with a strong strand displacement activity and high temperature tolerance ensures high amplification yield at constant temperature when working with impure or low-copy number targets as well as with complex templates. The Allin™ Bst Buffer includes optimal concentrations of magnesium and dNTPs, what minimizes pipetting steps. This ALLin™ Bst DNA Polymerase-buffer system together with a supplied enhancer, detects <5 DNA targets in a short time without the use of a thermocycler. This glycerol-free Bst DNA Polymerase is also an excellent tool for development of dry format lyophilized kits for pathogen detection using isothermal amplification techniques. For more convenience, we recommend our kit formulations: ALLin™ Isothermal DNA Amplification Kit and ALLin™ Isothermal 1Step RNA Amplification Kit, both including a master mix with optimized high-performance buffer. Technical characteristics of Bst DNA Polymerase large fragment: Strong 5’ - 3’ strand displacement activity 5’-3’ polymerase activity No 5’ - 3’ exonuclease activity No 3’ - 5’ exonuclease (proofreading) activity Minor reverse transcriptase activity (for RNA, use ALLin™ Isothermal 1Step RNA Amplification Kit (#IRK0101) which includes RTase) Optimal amplification temperature is 65°C. Working temperature range is 55-70°C. Optimal reaction time is 20 minutes, if needed, the reaction can be performed 30 to 60 minutes. The enzyme is inactivated in 10 minutes at 80°C. The use of this product in certain applications may be covered by patents. The user has to analyse all applicable Limited Use Label Licenses and may need licensing for certain cases.

The ALLin™ Hot Start Bst Polymerase is a recombinant protein, representing a large fragment of the B. stearothermophilus DNA Polymerase expressed in E. coli cells. The activity of the enzyme is blocked at ambient temperature due to molecular inhibition based hot-start technology, and the polymerase is activated only at 45-50°C what reduces non-specific amplification, primer dimer formation and is therefore excellent for ambient reaction setup. This robust polymerase with a strong strand displacement activity and high temperature tolerance ensures high amplification yield at constant temperature when working with impure or low-copy number targets as well as with complex templates. The Allin™ Bst Buffer includes optimal concentrations of magnesium and dNTPs, what minimizes pipetting steps. This ALLin™ Hot Start Bst Polymerase-buffer system together with a supplied enhancer, detects <3 DNA targets in a short time without the use of a thermocycler. This glycerol-free Hot Start Bst Polymerase is also an excellent tool for development of dry format lyophilized kits for pathogen detection using isothermal amplification techniques. For more convenience, we recommend our kit formulations: ALLin™ HS Isothermal DNA Amplification Kit and ALLin™ HS Iso-Colorimetric DNA Amplification Kit both including 2X master mix formulations. Technical characteristics of Hot Start Bst Polymerase (large fragment): ·  The polymerase is blocked at ambient temperature and activated at temperatures above 45°C. ·  Strong 5’ - 3’ strand displacement activity ·  Strong 5’ - 3’ polymerase activity ·  No 5’ - 3’ exonuclease activity ·  No 3’ - 5’ exonuclease (proofreading) activity ·  Only minor reverse transcriptase activity ·  Optimal amplification temperature is 65°C ·  Working temperature range is 55-70°C ·  Optimal reaction time is 20 minutes (depends on the buffer) ·  If needed, the reaction can be extended to 30 - 60 minutes ·  The enzyme is inactivated in 10 minutes at 80°C The use of this product in certain applications may be covered by patents of third parties. The user has to analyse all applicable Limited Use Label Licenses and may need licensing from third parties.  

ALLin™ HS Isothermal DNA Amplification Kit enables detection of as little as 3 target DNA molecules in a short time of 20 minutes even without the use of a thermal cycler. The Kit includes a 2X master mix with optimized high-performance buffer, dNTPs and a recombinant Hot Start Bst Polymerase having strong 5’ - 3’ strand displacement activity and efficient 5’ - 3’ polymerase activity working at 55 - 70°C. The activity of the enzyme is blocked at ambient temperature due to molecular inhibition based hot-start technology, and the polymerase is activated only at 45 - 50°C what reduces non-specific amplification, primer dimer formation and is therefore excellent for an ambient temperature reaction setup. The polymerase has neither 5’ - 3’ nor 3’ - 5’ exonuclease activity and retains only minor reverse transcription activity. The kit includes PCR water; only templates and primers have to be supplied by the user. Quantitative Fluorescent Dye is included for an optional use, for a real-time detection in FAM channel on any qPCR cycler.  ALLin™ HS Isothermal DNA Amplification Kit is a tool of choice for such applications like LAMP, WGA, RAM with an additional advantage of ambient temperature set up and higher temperature reactions, what makes amplification of complex and GC-rich templates more efficient.  Technical characteristics of Hot Start Bst Polymerase (large fragment): The polymerase is blocked at ambient temperature and activated at temperatures above 45°C. Strong 5’ - 3’ strand displacement activity  5’ - 3’ polymerase activity  No 5’ - 3’ exonuclease activity No 3’ - 5’ exonuclease (proofreading) activity  Minor reverse transcriptase activity (for RNA, use ALLin™ Isothermal 1Step RNA Amplification Kit (#IRK0101) which includes RTase) Optimal amplification temperature is 65°C.  Working temperature range is 55 - 70°C.  Optimal reaction time is 20 minutes, if needed, the reaction can be extended to 30 - 60 minutes The enzyme is inactivated in 10 minutes at 80°C.  Quantitative Fluorescent Dye has the excitation max. at 482 nm and emission max. at 512 nm. The use of this product in certain applications may be covered by patents. The user has to analyse all applicable Limited Use Label Licenses and may need licensing for certain cases.  

ALLin™ HS Iso-Colorimetric DNA Amplification Kit enables sensitive target DNA molecules detection in real time without the use of any equipment. The kit employs the robust Hot Start Bst polymerase in a master mix with all reaction components and the pH-sensitive inert dye which changes the colour from pale orange to bright yellow as soon as the reaction pH changes with the synthesis of DNA reaching a plateau. The mix detects as little as 3 target DNA molecules in a short time of 20 minutes without the use of any equipment. The 2X master mix includes the high-performance buffer, dNTPs and a recombinant Hot Start Bst Polymerase large fragment having strong 5’ - 3’ strand displacement activity and efficient 5’ - 3’ polymerase activity working at 55 - 70°C. The activity of the enzyme is blocked at ambient temperature due to molecular inhibition based hot-start technology, and the polymerase is activated only at 45 - 50°C what reduces non-specific amplification, primer dimer formation and is therefore excellent for ambient reaction setup. The polymerase has neither 5’ - 3’ nor 3’ - 5’ exonuclease activity and retains only minor reverse transcription activity. The kit includes PCR water; only templates and primers have to be supplied by the user ALLin™ HS Iso-Colorimetric DNA Amplification Kit is a tool of choice for such applications like LAMP, WGA, RAM with additional advantages of a room temperature set up, field detection as well as of high-temperature reactions, what makes amplification of complex and GC-rich templates more efficient. Technical characteristics of Hot Start Bst Polymerase (large fragment): The polymerase is blocked at ambient temperature and activated at temperatures above 45°C. Strong 5’ - 3’ strand displacement activity  5’ - 3’ polymerase activity  No 5’ - 3’ exonuclease activity No 3’ - 5’ exonuclease (proofreading) activity  Minor reverse transcriptase activity (for RNA, use ALLin™ Isothermal 1Step RNA Amplification Kit (#IRK0101) which includes RTase) Optimal amplification temperature is 65°C.  Working temperature range is 55 - 70°C.  Optimal reaction time is 20 minutes, if needed, the reaction can be extended to 30 - 60 minutes The enzyme is inactivated in 10 minutes at 80°C.  The use of this product in certain applications may be covered by patents. The user has to analyse all applicable Limited Use Label Licenses and may need licensing for certain cases.  

SampleIN™ Lysis Set for PCR/qPCR is a combination of a lysis buffer and protease-containing buffer allowing for a fast lysis of different sample material to release the DNA in a short time.The quickly lysed samples contain enough DNA for PCR and qPCR/RT-qPCR applications, eliminating the need of tedious template purification. The set is used to prepare templates for direct PCR or qPCR from mouse tail or ear, mammalian tissues, hair follicle, buccal swabs and blood. It can also be used with some plant materials, if these are not considered as very oily, hard or difficult to lyse. Rapid 15 min DNA extraction using DPK Lysis and Protease Buffers in a single tube generates crude PCR template extract which can be further amplified using inhibitor resistant, robust PCR or qPCR mixes such as ALLin™ HS Red Taq Mastermix, or SampleIN™ qPCR Probe Mixes.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. PCR / 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 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 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.Rapid 15 min DNA extraction using DPK Lysis and Protease Buffers in a single tube generates PCR template extract which is further amplified under fast cycling conditions with a hot-start Taq master mix that includes red dye for direct gel loading.In a 2% agarose TAE gel the red dye migrates with~350 bp DNA, in 1% agarose TAE gel with ~ 600 bp DNA fragments.The ALLin™ HS Red Taq Mastermix includes a hot start Taq DNA Polymerase what ensures high yield, specific, low background amplification. Mix components allow for a fast PCR cycling and increase success when working with complex templates or multiplexing. Generated A-tailed PCR products are suitable for ligating into TA cloning vectors, sequencing and other applications.

ALLin™ Isothermal DNA Amplification Kit enables detection of as little as 5 target DNA molecules in a short time of 20 minutes even without the use of a thermal cycler. The Kit includes a 2X master mix with optimized high-performance buffer, dNTPs and a recombinant Bst Polymerase large fragment having strong 5’ - 3’ strand displacement activity and efficient 5’-3’ polymerase activity working at 55-70°C. The Polymerase has neither 5’ - 3’ nor 3’ - 5’ exonuclease activity and retains only minor reverse transcription activity. The kit includes PCR water; only templates and primers have to be supplied by the user. Quantitative Fluorescent Dye, 20X is included for an optional use, for a real-time detection in FAM channel on any qPCR cycler. ALLin™ Isothermal DNA Amplification Kit is a tool of choice for such applications like LAMP, WGA, RAM with an additional advantage of higher temperature reactions, what makes amplification of complex and GC-rich templates more efficient. Technical characteristics of Bst DNA Polymerase large fragment: Strong 5’ - 3’ strand displacement activity 5’-3’ polymerase activity No 5’ - 3’ exonuclease activity No 3’ - 5’ exonuclease (proofreading) activity Minor reverse transcriptase activity (for RNA, use ALLin™ Isothermal 1Step RNA Amplification Kit (#IRK0101) which includes RTase) Optimal amplification temperature is 65°C. Working temperature range is 55-70°C. Optimal reaction time is 20 minutes, if needed, the reaction can be performed 30 to 60 minutes. The enzyme is inactivated in 10 minutes at 80°C. Quantitative Fluorescent Dye has the excitation max. at 482 nm and emission max. at 512 nm. The use of this product in certain applications may be covered by patents. The user has to analyse all applicable Limited Use Label Licenses and may need licensing for certain cases.

ALLin™ Isothermal 1Step RNA Amplification Kit enables detection of < 5 target RNA molecules in a short time of 30 minutes without the use of PCR cycler.  An RT5 Mix, the blend of a thermostable reverse transcriptase and highly efficient RNase Inhibitor is included in the kit to initially synthesize cDNA from RNA templates. The cDNA is amplified by Bst polymerase. Kit includes a 2X master mix with optimized high-performance buffer, dNTPs and a recombinant Bst Polymerase large fragment having strong 5’ - 3’ strand displacement activity and efficient 5’-3’ polymerase activity working at 55-70°C. The Polymerase has neither 5’ - 3’ nor 3’ - 5’ exonuclease activity and retains only minor reverse transcription activity. The kit also includes PCR water; only templates and primers have to be supplied by the user. Quantitative Fluorescent Dye, 20X is included for an optional use, for a real-time detection in FAM channel on any qPCR cycler. ALLin™ Isothermal 1Step RNA Amplification Kit is a tool of choice for isothermal amplification of RNA templates and such applications like RT LAMP, with an additional advantage of higher temperature reactions, what makes amplification of complex and GC rich templates more efficient. Technical characteristics of Bst DNA Polymerase large fragment: Strong 5’ - 3’ strand displacement activity 5’-3’ polymerase activity No 5’ - 3’ exonuclease activity No 3’ - 5’ exonuclease (proofreading) activity Minor reverse transcriptase activity Optimal amplification temperature is 65°C. Working temperature range is 55-70°C. The Bst enzyme is inactivated in 10 minutes at 80°C. RT5 Mix includes thermostable reverse transcriptase (modified MMuLV) working at high temperatures and a thermostable RNase inhibitor protecting template RNA from degradation. Optimal 1Step RT amplification reaction time is 30 minutes, if needed, the reaction can be performed 30 to 60 minutes. Quantitative Fluorescent Dye has the excitation max. at 482 nm and emission max. at 512 nm. The use of this product in certain applications may be covered by patents. The user has to analyse all applicable Limited Use Label Licenses and may need licensing for certain cases.

Recombinant phi29 DNA Polymerase is a classical enzyme dedicated for use in common isothermal DNA amplification applications that are carried out at moderate temperature based on a strand displacement activity. The enzyme is supplied with an optimized high-performance buffer. The user has to add dNTPs, template and primers. phi29 DNA Polymerase was discovered and characterized by M. Salas. The Polymerase has strong strand displacement activity and efficient 5’-3’ polymerase activity working at about 4-35°C and synthesizing DNA from minor amounts to enormous yield up to visibly increased the viscosity of the reaction mixture. The enzyme has no 5’-3’ exonuclease activity, but has strong 3’ - 5’ exonuclease (proofreading) activity, and may degrade primers, therefore the use of 3’ protected exo-resistant primers is recommended in the literature. The enzyme can be heat-inactivated, tolerates dUTP and produces blunt-ended DNA. Most important technical characteristics of phi29 DNA Polymerase (based on abundantly available scientific literature): Strong strand displacement activity 5’-3’ polymerase activity on DNA templates (and on RNA templates) Strong 3’ - 5’ (proofreading) exonuclease activity, may degrade unprotected primers No 5’ - 3’ exonuclease activity Optimal reaction temperature is 30°C Working temperature range is 4-35°C, depends on application Optimal reaction time depends on application The enzyme is inactivated in 10 minutes at 65°C. Can extend both DNA and RNA primers Addition of pyrophosphatase may accelerate the reaction The use of this product in certain applications, some additives or protocols may be covered by patents. The user has to analyse all applicable Limited Use Label Licenses and may need licensing for certain cases.