Universal Mastermix_RUO

The Universal Mastermix contains all the required reagents (except the primers and the DNA or RNA template) for the performance of LAMP analysis of DNA or RNA samples. The 2X Universal Mastermix is optimized to be used with the HNB dye, which is included in the kit, but it can also be used in combination with other colorimetric or fluorescent dyes, e.g. SYBR Green I or II. The Bst and Reverse transcriptase enzyme that are contained in the master mix can produce robust LAMP amplification in a very short running time. They can provide high sensitivity and specificity, allowing the specific amplification of DNA and RNA genomes and fragments, from low copy numbers. The HNB dye which is included in the kit allows LAMP colorimetric analysis of the samples. Using this dye a positive sample can be discriminated from a negative one by the naked eye or automatically in real-time when it is used with the PEBBLE-R qcLAMP platform which is optimized to be used with. It is very easy to handle, and the results are easily discriminated. Content The Universal Mastermix contains the enzyme mix with the Bst polymerase and the Reverse Transcriptase, dNTPs, buffers, nuclease-free water, the HNB dye, and mineral oil. Standard protocol The following protocol serves as a general guideline for the preparation of the master mix of all components except the template. Optimal reaction conditions (temperature, concentration of primers, and concentration of DNA or RNA template) may vary depending on the target. The optimal reaction volume for a single reaction is 25 μL. 1. Mix well and briefly centrifuge the 2X Universal Mastermix and the HNB dye after thawing. On ice prepare a mixture for the desired number of LAMP reactions 2.Mix well the reaction. The color of the mix should be purple/deep blue. 3. Add carefully 15 μL of mineral oil at the side of the vial and wait 30 sec until it forms a layer over the qcLAMP reaction. Make sure the oil is not mixed with the reaction mixture. 4. Incubate at 65oC for 30 minutes. The temperature and the time can be adjusted as necessary depending on the sequence of the primers and the concentration of the template. Addition of template The optimal amount of starting material may vary depending on the quality, stability, and the complexity of it. Both DNA and RNA templates can be used with the Universal Mastermix. It is recommended to use 10 copies or more of DNA or RNA templates.