highQu PCRbeam™ Fast PCR Detection Kit is a convenient tool for fast detection of gene-specific amplification products obtained by PCR, LAMP or RPA. The detection is based on immunological reaction driven by Biotin and FITC (fluorescein isothiocyanate), thus the amplified DNA shall include Biotin and FITC labels. The PCR amplification has to be performed with one primer labeled with FITC at 5’-end and one primer labeled with Biotin at 5’-end. Alternatively the use of one of the labeled primers can be replaced by gene-specific FITC or Biotin labeled probe. Kit includes PCRbeam™ Membrane Strips that are coated with biotin-ligand on the test band and an anti-rabbit antibody on the control band. The bottom part of the strip which is used for sample application contains an anti-FITC antibody attached to gold particles. PCRbeam™ Detection Buffer is Tris-buffered saline enabling the detection. The PCRbeam™ Fast PCR Detection Kit can be applied for established tests or home-brew assays as a fast and sensitive yes/no detection method. The detection sensitivity is up to 100 fold higher than the one achievable with ethidium bromide stained gels what provides an environment friendly save and economical alternative to the use of mutagen stains. For establishing sensitive PCR-based tests before PCRbeam™ detection we recommend the use of hot-start PCR enzymes or master mixes, like highQu ALLin™ Hot Start Taq Mastermix or ALLin™ Hot Start Taq DNA Polymerase. Principle: The membrane strip is soaked for 10 minutes into the vial with the detection buffer mixed with PCR product. The lateral sample flow driven by gold particles moves the solution up the strip. FITC labeled DNA strand binds with the anti-FITC antibody on the gold particle and Biotin labeled DNA strand is caught by Biotin ligand attached to the test band. As both DNA strands remain hybridized at room temperature, the test band builds an aggregate that develops red-blue color. Excess gold particles that were not caught by FITC move up the strip and the anti-FITC antibody binds to the anti-rabbit antibody to develop the red-blue colored control band. If there is no PCR product in the reaction, then only the control band will be visible. If there is a specific product, the test band will be colored as well.

High Resolution Melting analysis (HRM) is a fast and simple technique for identification of DNA sequence variations. It allows identifying single nucleotide differences by detecting minor changes in qPCR melting curves. highQu ORA™ HRM qPCR Mix includes a proprietary intercalating saturating dye showing no inhibition for PCR. The dye has the same affinity for both AT or GC rich sequences what leads to highest accuracy in genotyping. The hot-start function in the mix is based on the small molecular inhibitor technology and allows achieving highest sensitivity and specificity under both standard and fast qPCR cycling conditions. The mix provides excellent performance on both AT and GC rich templates and reliable results with minimum or no optimization. Our master mixes are supplied with PCR Water to guaranty the best performance.

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.

StainIN™ GREEN Nucleic Acid Stain is a significantly safer alternative to ethidium bromide. It is same easy to use, 4X as sensitive (under Blue LED) and much more secure. More economical as other green dyes, this stain can be also used and disposed with less environmental and health concerns compared to ethidium bromide. It is a fluorescent dye that allows detection of >0,1 ng of DNA in both agarose and polyacrylamide gels. It binds to both dsDNA, ssDNA and RNA and emits green fluorescence when bound to DNA and red fluorescence when bound to RNA detectable under the UV or Blue light and documented with same filters as similar green dyes. StainIN™ GREEN is ideal for DNA extraction from gels for cloning. Smaller than ethidium bromide carcinogenicity has been proved by Ames-test. Mammalian cell mutagenicity tests, both mouse marrow erythrocyte micronucleus and spermatocyte chromosomal aberration tests gave negative mutagenicity results. Carcinogenicity and toxicity of the widely used Ethidium Bromide (EtBr) is one of the biggest safety and environmental problems in the labs where gel electrophoresis is a daily routine. The less toxic nucleic acid dyes address this problem and minimize concerns. The StainIN™ GREEN (SYBR Green-like stains alternative), (SYBR® Green is a registered trade mark of Molecular Probes Inc.) and StainIN™ eco-RED Nucleic Acid Stains by highQu provide a much less-carcinogenic, significantly safer alternative to EtBr while delivering even several times higher detection sensitivity. Same like EtBr, these stains are used during the process of electrophoresis in an agarose gel or PAAG and in the electrophoresis running buffer. Nucleic acid stains are typically divided into two groups – they are either intercalating dyes or minor groove binders. The StainIN™ GREEN and StainIN™ eco-RED Nucleic Acid Stains belong to the group of intercalating dyes that bind to NA and produce the fluorescence signal. Same like many other suppliers of similar products, highQu retains the right to keep the precise chemical information undisclosed and proprietary. The StainIN™ GREEN Nucleic Acid Stain as well as the StainIN™ eco-RED Nucleic Acid Stain are both supplied in water. Detailed safety information is available in MSDS sheets of the stains. Despite proven less mutagenicity, all nucleic acid stains, including SYBR® Green or other shall be handled with care. Used electrophoresis buffer with a stain as well as melted gels shall be run through approved (carbon) filters. If the absence of residual fluorescence is confirmed, the liquids can be disposed with plenty of water down the drain. Alternatively, all used liquids can be autoclaved as the StainIN™ GREEN and StainIN™ eco-RED Nucleic Acid Stains degrade during the long exposure to above 100ºC temperature. The safety office shall be consulted periodically to match local regulations, as they vary and change.