RedMasterMix (2x) - PCR MasterMix with red dye

For reliable and easy PCR: PCR Master Mix for standard PCR applications (e.g. screenings) for amplifying DNA fragments <5kb. It is a premixed, ready-to-use solution containing Taq DNA Polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient amplification of DNA templates by routine PCR (one tube, one pipetting step). Just add your primers and template DNA - that´s all.  The special formulation of our PCR Master Mix guarantees you reproducible results even after repeated freezing/thawing steps leading to good yields and sensitivity. The PCR Master Mix is efficient (no left over of reagents), scalable from 10µL to 50µL and stable for 24 months. Test sample available at a special price! The test sample price will be refunded on the first official order of the product.   Our Agaroses > and DNA Ladders > are ideally suited for subsequent electrophoresis of PCR products.

RedMasterMix (2x) - PCR MasterMix with red dye to help visualize pipetting and mixing steps. Electrophoresis can be performed immediately after PCR without the need of a gel loading buffer. This makes this MasterMix time efficient, cost efficient and a reliable choice for the best PCR results with high efficiency.  One tube, one pipetting step. RedMasterMix (2x) is delivered to you as a optimized, ready-to-use solution containing- Taq DNA Polymerase - dNTPs- MgCl2- a red dye- reaction bufferfor efficient amplification of DNA. Additionally the RedMasterMix (2x) contains an additive and a red dye for proceeding with electrophoresis after PCR without adding loading buffer. Just add your primers and template DNA. This PCR MasterMix (2x) has been optimized for use in routine PCR amplification of DNA templates in the range of 0.2-4kb. The special formulation of our PCR RedMasterMix (2x) can withstand repeated freezing/thawing without compromising yields, sensitivity or results, when used as directed. The PCR MasterMix with red dye is efficient (no left over of reagents), scalable from 10µL to 50µL and stable for at least 24 months.Our RedMastermix can also be used for Sanger sequencing. Just dilute the PCR reaction 1:8 after PCR, or purify using spin columns and then apply. Read now in our blog how Genaxxon's RedMasterMix can also simplify your laboratory work. Our Agaroses > and DNA Ladders > are ideally suited for subsequent electrophoresis of PCR products.

Genaxxon’s FAST DNA polymerase (2X) MasterMix with dye is the master mix of our FAST DNA polymerase (M3285) which is a robust enzyme, ideally suited for applications like genotyping and screening, amplifying with extreme speed of up to 2 seconds per 1kb, as well as high yield and consistency. FAST DNA polymerase has 5’-3’ exonuclease activity, but no 3’-5’ exonuclease (proofreading) activity. PCR products generated with this enzyme are A-tailed and can thus be cloned into TA cloning vectors. The extreme speed of Genaxxon’s FAST DNA polymerase allows the use of an extension rate of 4-8 kb/min. Genaxxon bioscience’s FAST DNA Polymerase (2X) MasterMix with dye includes all components needed for a fast PCR, except the primers. Besides this, it includes an inert red tracking dye for gel electrophoresis. Therefore, upon completion of the PCR, the reaction is ready for direct loading onto an agarose gel without the need of adding an additional loading buffer.Example of a typical PCR protocol (graphical presentation)* Ta is the annealing temperature (Ta should be 2°C above Tm**)  ** Tm is the melting temperature of a primer which is defined as the temperature at which 50% of the primer bind to the complementary sequence of the target DNA. Please note: By increasing Ta above Tm, this percentage decreases, however, primers will still anneal up to a certain degree and initiate extension. Therefore, PCR would still work with a Ta of several degrees higher than the Tm but with a dramatically reduced efficiency. Hence, we recommend optimizing the Ta by performing a temperature gradient (e.g., starting at the lowest Tm or a few degrees below and increasing with 2°C increments). *** Largest tested amplicon size: 5kb. Please note that longer extension times may be required for targets larger than 5kb!

Ready-to-use PCR Mastermix with red Loading Dye for visual control of the pipetting steps and additional fluorescent dye for fast and easy detection of the DNA bands. After PCR, the PCR mix can be pipetted directly into the gel pockets without adding loading buffer. This makes our RedMasterMix Fluoro (2x) even more time- and cost-saving than our proven Red Mastermix. The RedMasterMix Fluoro (2x) with fluorescent gel staining dye and red loading dye also features high specificity for best results. The RedMasterMix Fluoro (2x) is a ready-to-use mixture of: - Taq DNA Polymerase- PCR reaction buffer- dNTPs- MgCl2- red loading dye- fluorescence gel staining dye for DNA band detection in an optimal concentration for efficient amplification of DNA templates by PCR. Only the primers and the template DNA have to be added. At the same time, the PCR Mastermix contains an additive and a red dye, which allows subsequent electrophoresis without the addition of loading buffer. After electrophoresis, detection is performed directly under blue light (ca. 490nm) without further staining. This saves additional time and costs. The RedMasterMix Fluoro (2x) with fluorescent dye was developed for use in routine PCR up to 4 kb amplicon length. The special composition of the buffer guarantees reproducible results even after repeated thawing and freezing cycles. Our Red MasterMix (2x) Fluoro is shipped in convenient aliquots of 1.25mL.Please note: The included fluorescent dye does show only a small excitation peak in the UV range, but fluorescence is significantly stronger under blue light excitation at 490nm. We therefore recommend using blue light to achieve the best possible results.

Highly specific Hotstart master mix for routine and diagnostic PCR and qPCR up to 4kb: ready-to-use master mix with chemical modified Taq DNA polymerase. This Hotstart Master Mix for qPCR is a premixed, ready-to-use solution containing chemically modified Taq DNA Polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient amplification of DNA templates by PCR and qPCR (one tube, one pipetting step). Just add your primers and template DNA. The Hotstart PCR Master Mix has been optimized for use in routine and diagnostic Hotstart PCR and qPCR for amplifying DNA template in the range of 0.2-4kb. The special formulation of our Hotstart Master Mix guarantees you reproducible results even after repeated freezing/thawing steps leading to good yields and sensitivity. The Hotstart Master Mix is efficient (no left over of reagents), scalable from 10µL to 50µL and stable for 12 months. This Hotstart PCR master mix is also available as SuperHot Mastermix Blue > enabling visualisation of each pipetting step. With our high quality dNTPs as Set (M3015.4100 and M3015.0250) > or Mix (M3016.1010) > or our DNA Ladders > and favourable standard agarose (M3044) > we can offer additional products for your PCR.

Genaxxon’s FAST HotStart DNA polymerase (2X) MasterMix with dye is the master mix of our FAST HotStart DNA polymerase (M3287) which is a robust enzyme, ideally suited for applications like genotyping and screening, amplifying with extreme speed of up to 2 seconds per 1kb, as well as high yield and consistency.PCR products generated with this enzyme are A-tailed and can thus be cloned into TA cloning vectors. The extreme speed of Genaxxon’s FAST HotStart DNA polymerase allows the use of an extension rate of 4-8 kb/min. The buffer composition has been optimized via high-throughput screening and makes the enzyme particularly resistant to PCR inhibitors. Due to its robustness, Genaxxon’s FAST HotStart DNA polymerase (2X) MasterMix with dye is suitable for direct PCR from unpurified samples, including blood, urine, and bacterial colonies, making it the ideal choice for consistent results in fast complex PCR amplifications. Besides this, it includes an inert red tracking dye for gel electrophoresis. Therefore, upon completion of the PCR, the reaction is ready for direct loading onto an agarose gel without the need of adding an additional loading buffer.Example of a typical PCR protocol (graphical presentation)* Ta is the annealing temperature (Ta should be 2°C above Tm**)  ** Tm is the melting temperature of a primer which is defined as the temperature at which 50% of the primer bind to the complementary sequence of the target DNA. Please note: By increasing Ta above Tm, this percentage decreases, however, primers will still anneal up to a certain degree and initiate extension. Therefore, PCR would still work with a Ta of several degrees higher than the Tm but with a dramatically reduced efficiency. Hence, we recommend optimizing the Ta by performing a temperature gradient (e.g., starting at the lowest Tm or a few degrees below and increasing with 2°C increments). *** Largest tested amplicon size: 5kb. Please note that longer extension times may be required for targets larger than 5kb!

The RedMastermix Hot (2x) from Genaxxon for fast and easy PCR setup and high reproducibility in standardized applications (Colony PCR, High-throughput PCR, Automated PCR applications). The Mastermix contains a red dye that allows direct application of the PCR product to the gel. The red dye enables monitoring of the gel run. Compared to standard Taq PCR mastermixes, the hotstart polymerase and the optimized buffer mixture with MgCl2 and dNTP results in an increased yield of amplicons up to 6kb even under Fast PCR conditions. Highly specific Hotstart Mastermix for routine applications. FAST PCR amplification up to 2kb. Ready-to-use, consisting of: Antibody inhibited Taq DNA Polymerase optimized PCR-Puffer dNTPs MgCl2 for your HotStart PCR. Ready-to-use Hotstart Mastermix. All you have to do is add the primers and the template DNA. The special composition of the buffer guarantees reproducible results even after repeated thawing and freezing cycles. The Hotstart Mastermix will be sent in 1mL aliquots. With our high quality dNTPs as Set (M3015.4100 and M3015.0250) > or Mix (M3016.1010) > or our DNA Ladders > and favourable standard agarose (M3044) > we can offer additional products for your PCR.

The Genaxxon NGSClone PCR Mastermix (2x) combines high-fidelity DNA polymerase with antibody-mediated Hot-Start technology for precise, fast, and reliable PCR results. Perfect for challenging templates such as GC-rich DNA, cDNA, or complex plant and animal samples. This ready-to-use master mix contains all essential components – just add primers and template – making it ideal for high-fidelity PCR and NGS library preparation.NGSClone PCR Mastermix (2x) – High-Fidelity & Hot-Start for Accurate PCRMaximum accuracy for demanding PCR applicationsThe Genaxxon NGSClone PCR Mastermix (2x) delivers outstanding performance by combining a novel high-fidelity DNA polymerase with antibody-mediated Hot-Start technology in a convenient ready-to-use PCR master mix. With up to 120× higher fidelity compared to standard Taq polymerases, it ensures highly accurate amplification even for GC-rich templates. Ideal for PCR in NGS workflows, cDNA amplification, and plant or animal DNA analysis.Key Features of the NGSClone PCR Mastermix (2x)Exceptional accuracy – High-fidelity DNA polymerase with strong 3’-5’ exonuclease activity (proofreading) for error-free amplification Fast & efficient PCR – Engineered enzyme structure with enhanced domain for high processivity and faster extension rates Fast amplification – achieves target amplification in just 10–15 seconds per kbHot-Start precision – Antibody blocking keeps the enzyme inactive at room temperature, preventing non-specific amplification and improving specificity Broad applicability – Suitable for GC-rich DNA, cDNA, plant and animal templates, and other difficult targets Easy workflow – Contains polymerase, dNTPs, and optimized buffer; just add primers and DNA Benefits in Your Lab Saves time and reduces pipetting stepsMinimizes cross-contamination Delivers high reproducibility Remains stable after repeated freeze-thaw cycles Applications High-fidelity PCR for error-free cloning PCR of GC-rich templates cDNA amplification NGS library preparation Standard PCR requiring high accuracy Genaxxon’s NGSClone PCR Mastermix (2x) – for fast, precise, and reproducible PCR results with maximum convenience.The Genaxxon NGSClone PCR Mastermix Red (2x) is also available with a red loading dye.

AQ97 Hot Start High Fidelity DNA Polymerase ist eine proofreading Polymerase mit hoher Amplifikationsgeschwindigkeit und sehr hoher Genauigkeit (>60-fach) gegenüber Taq DNA Polymerase. AQ97 ist ideal für schwierige Targets mit sehr niedrigem oder hohem GC-Gehalt geeignet. Neben diesen Eigenschaften zeichnet sich die AQ97 auch durch Ihre Eignung, lange Targets bis zu 18 kb, zu amplifizieren aus. Dies führt zu sehr genauen und zuverlässigen PCR-Ergebnissen.  AQ97 High Fidelity DNA Polymerase besitzt sowohl eine 5'→3' DNA Polymerase-Aktivität als auch eine 3'→5' proofreading Exonuklease-Aktivität, die es dieser Polymerase ermöglicht, Basenpaar-Fehlpaarungen zu korrigieren. AQ97 High Fidelity DNA Polymerase ist ein fusionierter Proteinkomplex aus DNA-Polymerase mit einer prozessivitätssteigernden DNA-Bindungsdomäne. Neben einer sehr schnellen und robusten Amplifikation komplexer und langer Zielmoleküle zeichnet sich die AQ97 High Fidelity DNA Polymerase durch eine hohe Genauigkeit aus, was dazu führt, dass auch für lange Amplicons eine genaue Amplifikation gewährleistet ist. Dadurch eignet sich die AQ97 besonders für PCR-Experimente, die eine Amplifikation mit sehr niedrigen Fehlerraten erfordern, wie z. B. Klonen/Subklonen, NGS-Anwendungen, SNP-Analysen und Mutagenese. Eigenschaften: • High Fidelity: >60x Taq fidelity• High elongation rate: 10 sec/kb (bis 6000bp per minute) • Built in hot start technology• Long range amplification: 18 kb for human gDNA• 5'-3' polymerase acitivity and 3'-5' exonuclease activity.• generates blund ends. Bild 1: Die Vergleichswerte der Genauigkeitswerte für die AQ97-DNA-Polymerase, die AccuPol-DNA-Polymerase, die beiden Wettbewerber High-Fidelity-DNA-Polymerasen "P" und "Q" und die Taq-DNA-Polymerase wurden durch eine NGS-basierte Analyse des Nukleotid-Fehleinbaus während der PCR ermittelt. Die PCR-Amplifikation wurde mit einem synthetischen DNA-Target von etwa 200 bp durchgeführt, wobei für jede der getesteten Polymerasen PCR-Produkte erzeugt wurden (unter Verwendung der empfohlenen Setup-Bedingungen). Bild 2: Acht verschiedene humane genomische DNA-Targets mit einer Länge von 400 bis 800 bp und einem GC-Gehalt zwischen 29% und 78% wurden mit dem AQ97 Hot Start High Fidelity DNA Polymerase 2x Master Mix amplifiziert. Für alle Targets wurde eine robuste Amplifikation beobachtet. Für Targets mit einem GC-Gehalt von mehr als 70% wurde der Reaktionsmischung 2 M Betain Enhancer Solution zugesetzt. Marker: M.  Bild 3: Es wurden fünf verschiedene Targets aus menschlicher genomischer DNA mit einer Länge von 2 kb bis 17,5 kb amplifiziert. Für alle Targets wurde eine robuste Amplifikation mit dem AQ97 Hot Start High Fidelity DNA Polymerase 2x Master Mix beobachtet, was die Fähigkeit des Master Mixes zur Amplifikation großer und komplexer Targets belegt. Die Amplikongrößen sind am oberen Rand des Gels angegeben. Marker M: High Range DNA-Leiter.

The SNP Pol DNA polymerase > used for the SNP Pol 2X Mastermix has been specially designed for easy, reliable and rapid allele-specific discrimination, eg. CRISPR / Cas9 point mutations, for detecting incorrect CRISPR / Cas9 products, or validating sequencing results. The SNP Pol DNA polymerase distinguishes highly specific, whether a mismatch of the primer-template-complex is present or not. The mismatch (point mutation) must be at the 3 'end of the primer. Thus, mutant alleles can be distinguished exactly from wild-type alleles - without sequencing, since the polymerase simply does not amplify in the case of a mismatch. Just place your primer on the supposed point mutation (Important: The point mutation must be at the 3 'end) and the polymerase will detect a mismatch in this region with almost 100% accuracy: If the template base complementary to the 3' end of the primer shows the mutation and the primer does not, no amplification takes place - 100% certainty within a short time!The SNP Pol DNA polymerase can therefore be easily, time and cost-effectively used for the screening of point mutations. The variant SNP PolTaq DNA polymerase has 5'-3 'nuclease activity and can therefore be used for specific hydrolysi probes such as Taqman® probes or Molecular beacons. For more information on our SNP DNA polymerases and applications, read our blog now. DescriptionSNP Pol DNA Polymerase and SNP PolTaq Polymerase are  highly selective DNA polymerase for the detection of Single Nucleotide Polymorphisms. It was developed specifically for allele-specific discrimination, where a very high discrimination rate is required e.g., in allele-specific PCR (ASA; AS-PCR), allele-specific primer extension (AS-PEX), SNP analysis, genotyping or in methylation-specific PCRs (MSP). Many other DNA polymerases tolerate mismatched primer-template complexes and are therefor not suitable. The SNP Pol DNA polymerase, on the other hand, distinguishes these specifically (high discrimination) and supplies only PCR products with perfectly matching primer pairs! The Genaxxon SNP Pol and SNP PolTaq DNA polymerases differ up to 100% by means of allele-specific PCR between the two alleles and, after a simple qPCR, give a clear result as to which allele is present. Thus, the principly great potential of the CRISPR/Cas9 technology can be used for human medicine and plant biotechnology especially together with the SNP PolTaq or SNP Pol DNA polymerase. Allele-specific PCR can be used to quantify the mutation rate in a pool or background of wild-type sequences. The verification of mutation frequencies determined by NGS can also be verified by means of allele-specific PCR and SNP Pol DNA polymerase. The SNP PolTaq DNA polymerase is very suitable for the analysis of liquid biopsy samples. With SNP PolTaq, the presence and frequency of cancer mutations can be analyzed and quantified very well. Picture below: Application note SNP Pol DNA Polymerase   We recommend short amplicon length (about 60-200 bp) for best results, but also longer amplicon lengths are possible. The addition of additional Magnesium (+0.5 - 1.5mM) might be needed in case of longer amplicons >500 bp.Trouble shooting: No bands after PCR of 30 cycles! Optimisation procedure for missing or weak bands.Annealing temperature is too low! Attention: The SNP Pol is an aptamer inhibited DNA polymerase. The aptamer oligo used reversibly inhibits the polymerase at temperatures <55°C! Therefore, the primers should ideally have an annealing temperature of >57°C. - realtime PCR approach - Check the dNTP concentration. This should be between 200 and 300µM (in the PCR). - Increase the number of cycles (at least 35, preferably equal to 40) Test optimisation - number of cyclesIn endpoint detection, a cycle count of 30 is not always sufficient to generate a clearly visible band. For example, with less than 200 DNA copies as a starting value. The test should therefore be run using real-time PCR, or five parallel PCRs should be used, one of which is taken from the PCR device after each of five different cycles (example below). Endpoint detection: Set up five identical PCR reactions in parallel using the SNP Pol DNA polymerase. Remove one of the PCR tubes from the cycler at each of 20, 25, 30, 35 and 40 cycles and apply all five reactions to an agarose gel at the end. This makes it easy to recognise which is the optimum number of cycles in the PCR for the given application (starting material, target, primer). SNP Pol PCR with cell lysates Animal cells and E.coli can be used directly for PCR with the SNP Pol DNA polymerase! No separate lysis or proteinase K digestion is necessary. PCR procedure:1. 50 - 500 cells are required per PCR batch! 2. prepare PCR mix with primers and buffer and place on ice! 3. add the picked colony directly to 10-20µL water (no separate lysis and no proteinase K digestion), vortex briefly (5 seconds), then add this cell suspension directly to the PCR reaction mixture, e.g. 10µL cell suspension for a PCR preparation with a total volume of 20µL. An initial denaturation time of 2-3 minutes is sufficient is sufficient, can be extended to 5 minutes if necessary. Genomic DNA per reaction max. 100 copies is relatively low, but should still work. The remainder of this cell suspension can also be frozen away in order to carry out further tests. Optional: 4. if possible: do real-time PCR!The SNP Pol DNA polymerase (M3009 > or M3061 >) can be used together with non-specific fluorescent dyes (eg Genaxxon's Green DNA Dye > or SybrGreen®) in real-time PCR. When working with specific PCR probes, only the SNP PolTaq DNA polymerase can be used, since only these have a 5'-3 'exonuclease activity. With our high quality dNTPs as Set (M3015.4100) > or Mix (M3016.1010) > or our DNALadders > and our favourable standard agarose (M3044) > we can offer additional products for your PCR. Application areas for SNP Pol DNA and SNP Pol DNA polymerase- Monitoring, verification and detection of point mutations- Identification of correct or wrong CRISPR/Cas9 products- Verification/validation of sequencing results- Quantification of mutations (e.g. NGS results)- SNP-detection by allele-specific amplification (ASA) / Allele-specific PCR- Methylation specific PCRs (MSP) after bisulfite treated DNA (CpG methylation sides)- HLA genotyping- micro sequencing- realtime PCR with hydrolysis probes- realtime multiplex PCRs - DamID-seq data in C. elegans. As an alternative to ChIP, DNA adenine methyltransferase identification by sequencing (DamID-seq) was recently shown to be able to characterize binding sites in single mammalian cells. Additionally, DamID can be achieved for cell-type specific analysis by expressing Dam fusion proteins under tissue specific promoters in a controlled manner. In this report, we present a user-friendly pipeline to analyse DamID-seq data in C. elegans. Sharma R, Ritler D, Meister P.Tools for DNA adenine methyltransferase identification analysis of nuclear organization during C. elegans development. Genesis. 2016 Feb 4. doi: 10.1002/dvg.22925. - Minisequencing SNP genotyping with SNPase DNA Polymerase can be carried out by the procedure described in: Lovmar L, Fredriksson M, Liljedahl U, Sigurdsson S, Syvänen AC. Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA. Nucleic Acids Res. 2003;31:e129. - Allel specific mismatch selectivity by the HiDi DNA polymerase Drum M, Kranaster R, Ewald C, Blasczyk R, Marx A (2014) Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification. PLoS ONE 9(5): e96640. doi:10.1371/journal.pone.0096640

The SNP PolTaq DNA polymerase used for the SNP PolTaq 2X Master Mix has been specially designed for easy, reliable and rapid allele-specific discrimination, eg. CRISPR / Cas9 point mutations, for detecting incorrect CRISPR / Cas9 products, or validating sequencing results. The SNP PolTaq DNA polymerase distinguishes highly specific, whether a mismatch of the primer-template-complex is present or not. The mismatch (point mutation) must be at the 3 'end of the primer. Thus, mutant alleles can be distinguished exactly from wild-type alleles - without sequencing, since the polymerase simply does not amplify in the case of a mismatch. Just place your primer on the supposed point mutation (Important: The point mutation must be at the 3 'end) and the polymerase will detect a mismatch in this region with almost 100% accuracy: If the template base complementary to the 3' end of the primer shows the mutation and the primer does not, no amplification takes place - 100% certainty within a short time!The SNP PolTaq DNA polymerase can therefore be easily, time and cost-effectively used for the screening of point mutations. The SNP PolTaq DNA polymerase has 5'-3 'nuclease activity and can therefore be used for specific hydrolysi probes such as Taqman® probes or Molecular beacons. For more information on our SNP DNA polymerases and applications, read our blog now. DescriptionSNP PolTaq DNA polymerase is a highly selective DNA polymerase for the detection of Single Nucleotide Polymorphisms. It was developed specifically for allele-specific discrimination, where a very high discrimination rate is required e.g., in allele-specific PCR (ASA; AS-PCR), allele-specific primer extension (AS-PEX), SNP analysis, genotyping or in methylation-specific PCRs (MSP). Many other DNA polymerases tolerate mismatched primer-template complexes and are therefor not suitable. The SNP PolTaq DNA polymerase, on the other hand, distinguishes these specifically (high discrimination) and supplies only PCR products with perfectly matching primer pairs! The Genaxxon SNP Pol and SNP PolTaq DNA polymerases differ up to 100% by means of allele-specific PCR between the two alleles and, after a simple qPCR, give a clear result as to which allele is present. Thus, the principly great potential of the CRISPR/Cas9 technology can be used for human medicine and plant biotechnology especially together with the SNP PolTaq or SNP Pol DNA polymerase. Allele-specific PCR can be used to quantify the mutation rate in a pool or background of wild-type sequences. The verification of mutation frequencies determined by NGS can also be verified by means of allele-specific PCR and SNP Pol DNA polymerase. The SNP PolTaq DNA polymerase is very suitable for the analysis of liquid biopsy samples. With SNP PolTaq, the presence and frequency of cancer mutations can be analyzed and quantified very well. Picture below: Application note SNP Pol DNA Polymerase   We recommend short amplicon length (about 60-200 bp) for best results, but also longer amplicon lengths are possible. The addition of additional Magnesium (+0.5 - 1.5mM) might be needed in case of longer amplicons >500 bp.Trouble shooting: No bands after PCR of 30 cycles! Optimisation procedure for missing or weak bands.Annealing temperature is too low! Attention: The SNP PolTaq is an aptamer inhibited DNA polymerase. The aptamer oligo used reversibly inhibits the polymerase at temperatures <55°C! Therefore, the primers should ideally have an annealing temperature of >57°C. - realtime PCR approach - Check the dNTP concentration. This should be between 200 and 300µM (in the PCR). - Increase the number of cycles (at least 35, preferably equal to 40) Test optimisation - number of cyclesIn endpoint detection, a cycle count of 30 is not always sufficient to generate a clearly visible band. For example, with less than 200 DNA copies as a starting value. The test should therefore be run using real-time PCR, or five parallel PCRs should be used, one of which is taken from the PCR device after each of five different cycles (example below). Endpoint detection: Set up five identical PCR reactions in parallel using the SNP Pol DNA polymerase. Remove one of the PCR tubes from the cycler at each of 20, 25, 30, 35 and 40 cycles and apply all five reactions to an agarose gel at the end. This makes it easy to recognise which is the optimum number of cycles in the PCR for the given application (starting material, target, primer). SNP Pol PCR with cell lysates Animal cells and E.coli can be used directly for PCR with the SNP Pol DNA polymerase! No separate lysis or proteinase K digestion is necessary. PCR procedure:1. 50 - 500 cells are required per PCR batch! 2. prepare PCR mix with primers and buffer and place on ice! 3. add the picked colony directly to 10-20µL water (no separate lysis and no proteinase K digestion), vortex briefly (5 seconds), then add this cell suspension directly to the PCR reaction mixture, e.g. 10µL cell suspension for a PCR preparation with a total volume of 20µL. An initial denaturation time of 2-3 minutes is sufficient is sufficient, can be extended to 5 minutes if necessary. Genomic DNA per reaction max. 100 copies is relatively low, but should still work. The remainder of this cell suspension can also be frozen away in order to carry out further tests. Optional: 4. if possible: do real-time PCR!The SNP Pol DNA polymerase (M3009 > or M3061 >) can be used together with non-specific fluorescent dyes (eg Genaxxon's Green DNA Dye > or SybrGreen®) in real-time PCR. When working with specific PCR probes, only the SNP PolTaq DNA polymerase can be used, since only these have a 5'-3 'exonuclease activity. With our high quality dNTPs as Set (M3015.4100) > or Mix (M3016.1010) > or our DNALadders > and our favourable standard agarose (M3044) > we can offer additional products for your PCR. Application areas for SNP Pol DNA and SNP PolTaq DNA polymerase- Monitoring, verification and detection of point mutations- Identification of correct or wrong CRISPR/Cas9 products- Verification/validation of sequencing results- Quantification of mutations (e.g. NGS results)- SNP-detection by allele-specific amplification (ASA) / Allele-specific PCR- Methylation specific PCRs (MSP) after bisulfite treated DNA (CpG methylation sides)- HLA genotyping- micro sequencing- realtime PCR with hydrolysis probes- realtime multiplex PCRs - DamID-seq data in C. elegans. As an alternative to ChIP, DNA adenine methyltransferase identification by sequencing (DamID-seq) was recently shown to be able to characterize binding sites in single mammalian cells. Additionally, DamID can be achieved for cell-type specific analysis by expressing Dam fusion proteins under tissue specific promoters in a controlled manner. In this report, we present a user-friendly pipeline to analyse DamID-seq data in C. elegans. Sharma R, Ritler D, Meister P.Tools for DNA adenine methyltransferase identification analysis of nuclear organization during C. elegans development. Genesis. 2016 Feb 4. doi: 10.1002/dvg.22925. - Minisequencing SNP genotyping with SNPase DNA Polymerase can be carried out by the procedure described in: Lovmar L, Fredriksson M, Liljedahl U, Sigurdsson S, Syvänen AC. Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA. Nucleic Acids Res. 2003;31:e129. - Allel specific mismatch selectivity by the HiDi DNA polymerase Drum M, Kranaster R, Ewald C, Blasczyk R, Marx A (2014) Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification. PLoS ONE 9(5): e96640. doi:10.1371/journal.pone.0096640

AQ97 High Fidelity DNA Polymerase ist eine proofreading Polymerase mit hoher Amplifikationsgeschwindigkeit und sehr hoher Genauigkeit (>60-fach) gegenüber Taq DNA Polymerase. AQ97 ist ideal für schwierige Targets mit sehr niedrigem oder hohem GC-Gehalt geeignet. Neben diesen Eigenschaften zeichnet sich die AQ97 auch durch Ihre Eignung, lange Targets bis zu 18 kb, zu amplifizieren aus. Dies führt zu sehr genauen und zuverlässigen PCR-Ergebnissen.  AQ97 High Fidelity DNA Polymerase besitzt sowohl eine 5'→3' DNA Polymerase-Aktivität als auch eine 3'→5' proofreading Exonuklease-Aktivität, die es dieser Polymerase ermöglicht, Basenpaar-Fehlpaarungen zu korrigieren. AQ97 High Fidelity DNA Polymerase ist ein fusionierter Proteinkomplex aus DNA-Polymerase mit einer prozessivitätssteigernden DNA-Bindungsdomäne. Neben einer sehr schnellen und robusten Amplifikation komplexer und langer Zielmoleküle zeichnet sich die AQ97 High Fidelity DNA Polymerase durch eine hohe Genauigkeit aus, was dazu führt, dass auch für lange Amplicons eine genaue Amplifikation gewährleistet ist. Dadurch eignet sich die AQ97 besonders für PCR-Experimente, die eine Amplifikation mit sehr niedrigen Fehlerraten erfordern, wie z. B. Klonen/Subklonen, NGS-Anwendungen, SNP-Analysen und Mutagenese. Eigenschaften: • High Fidelity: >60x Taq fidelity• High elongation rate: 10 sec/kb (bis 6000bp pro Minute)• Long range amplification: 18 kb for human gDNA• 5'-3' polymerase acitivity and 3'-5' exonuclease activity.• generates blund ends.Picture 1: Comparison figures of fidelity values for AQ97 DNA Polymerase, AccuPol DNA Polymerase, two well-recognized high fidelity DNA polymerases P and Q and Taq DNA Polymerase were determined through NGS-based analysis of nucleotide misincorporation during PCR. Initially, PCR amplification was performed on a ~ 200 bp synthetic DNA target, generating PCR products for each of the tested polymerases (using recommended setup conditions).  Picture 2: Distribution of substitution errors. PCR was performed using Taq DNA Polymerase, AQ97 High Fidelity DNA Polymerase, high fidelity DNA Polymerase Q and high fidelity DNA Polymerase P. The PCR was followed by NGS sequencing of the PCR products. The number of substitutions at each PCR target position was calculated and plotted in diagram A. Substitutions include misincorporated nucleotides and deletions at each position. Non-polymerase errors are subtracted from the total number of errors to revel true polymerase errors. Non-polymerase errors include mutations caused by thermocycling-induced DNA damage, pre-NGS sample preparation and sequencing errors. In these diagrams the average number of substitutions for Taq DNA Polymerase (Taq average) and for AQ97 High Fidelity DNA Polymerase (AQ97 average) is also plotted. Diagram B magnifies the area near the detection limit, displaying more information about the number of substitutions for AQ97 High Fidelity DNA Polymerase, high fidelity DNA Polymerase Q and high fidelity DNA polymerase P. Click to enlarge diagrams.