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.

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.

highQu ALLin™ Hot Start Taq DNA Polymerase is the superior sensitive enzyme. The activity at room temperature is blocked by small molecular inhibitor. Enzyme becomes active only after heating what allows for highly specific and extremely sensitive amplification, no primer dimer formation and no background. In combination with the optimized ALLin™ buffer enzyme provides higher success rates in demanding PCR applications like amplification of crude, complex or longer templates and fast cycling. ALLin™ Hot Start Taq DNA Polymerase has the same PCR accuracy like Taq DNA Polymerase, 4.5 x 10^4 (nucleotides incorporated before the error occurs) and produces A-tailed products suitable for ligating into TA cloning vectors. The convenience of ALLin™ Hot Start Taq DNA Polymerase is maximized by the use of 2X Red colored Mastermix providing the additional advantage of reduced pipetting and minimized errors. The mastermix is even supplied with PCR Water, and the only thing to add is the template with primers. ALLin™ HS 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. ALLin™ HS Red Taq Mastermix, 2X is also a key component in highQu SampleIN™ Direct PCR Kit (DPK0101/5), ensuring outstanding PCR results with crude samples.

highQu 1Step RT PCR Kit combines the 20X RT and RNase Inhibitor mix for efficient reverse transcription and the 2X PCR Mastermix for subsequent amplification of cDNA in the same tube. RT2 Mix, 20X is a blend of the engineered MMuLV (stable at 40-55°C allowing for high yields of long transcripts) with an efficient Ribonuclease Inhibitor protecting the template RNA from RNases. The resulting cDNA is then amplified by the 1Step RT PCR Mastermix, 2X. The PCR mastermix contains our proprietary Hot Start Taq DNA Polymerase. The enzyme activity at room temperature is blocked by small molecular inhibitor. Enzyme becomes active only after heating what allows for highly specific and extremely sensitive amplification, no primer dimer formation and no background. In combination with the optimized buffer the enzyme provides higher success rates in demanding PCR applications like amplification of complex or longer templates and fast cycling. Hot Start Taq DNA Polymerase has the same PCR accuracy like Taq DNA Polymerase, 4.5 x 104 (nucleotides incorporated before the error occurs), and produces A-tailed products suitable for ligating into TA cloning vectors. For the maximum convenience the Kit includes even the PCR Water to set up the reaction, so the only thing you need to take care of is the high quality RNA template.