Description Biofargo Fluorescent RT-LAMP Kit is designed to provide a one-step solution for Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) of RNA and an examination of the amplification results with real-time fluorescent detectors like real-time PCR machines. It has the following features： Optimized RT-LAMP mix, contains a fluorescent dye. Real-time monitoring of the RT-LAMP reactions using real-time PCR machine. Useful in optimizing RT-LAMP primers or reaction parameters. 4× concentration RT-LAMP mix allows larger in-put sample volumes than 2× concentration mix, which can increase the sensitivity of the test. High amplification efficiency, generally an order of magnitude more sensitive than RT-PCR. Isothermal amplification around 65°C, no need for expensive thermal cycling instruments. Template-primer mix supplied as RT-LAMP positive control, eliminates false negative results. CAT#211210-50 is sufficient for 50 reactions of 20μL volume, and CAT#211210-500 for 500 reactions of 20μL volume. Input Sample Requirements Perform DNA purification with a positive and a negative control. For a sample number of N, perform N+2 purifications. For the two additional purifications, one is for positive control and the other is for negative control. Kit Contents Component CAT#:211210-50 CAT#:211210-500 Fluorescent RT-LAMP Mix 200μL 1mL RTase-Bst Mix 100μL 500μL Template-primer Mix 50μL 250μL Storage and Handling Store the kit at -20°C. The kit is stable for at least one year from date of receipt. Required Materials not Supplied No Item 1 Prepared RNA Samples 2 RT-LAMP primers targeting RNA samples 3 Reaction vessels compatible with instrument for RT-LAMP 4 Molecular biology grade ddH2O 5 Real-time PCR machine Procedural Guidelines Perform all steps at room temperature (20–25°C) unless otherwise noted. Regularly de-contaminate working areas and equipment using chlorine bleach to avoid potential carryover contamination. Avoid opening reaction vessels after a RT-LAMP reaction is completed. Screening at least 10 sets of RT-LAMP primers for optimal sensitivity and specificity before choosing a final set. Use Loop primers if possible. Primers should be HPLC purified. Procedures Make 20×Primer Mix. Use appropriate amount of ddH2O to dissolve the HPLC-purified RT-LAMP primers at 100μM each. Use the 100μM stocks to make 100μL 20×Primer Mix according to the following table. If volumes other than 100μL (50μL, 200μL etc.) is desired, adjust volumes accordingly. Components to add Volumeto add Final Concentrationin 20×Primer Mix 100μM FIP 32μL 32 μM 100μM BIP 32μL 32 μM 100μM Loop F 4μL 8 μM 100μM Loop B 4μL 8 μM 100μM F3 2μL 4 μM 100μM B3 2μL 4 μM ddH2O 24μL - Thaw all components to be used at room temperature. Vortex briefly Fluorescent RT-LAMP Mix and Template-primer Mix. Centrifuge to collect material and place on ice. Don’t vortex RTase-Bst Mix. When using the kit for the first time，add all RTase-Bst Mix to Fluorescent RT-LAMP Mix and gently invert the tube for one minute to mix thoroughly. For a purification number of N+2, set up N+4 RT-LAMP reactions as described below. Volumes listed are for 20μL reaction. For the two additional reactions, one is for RT-LAMP Positive Control (PC) and the other is for RT-LAMP Negative Control (NC). PC NC N+2 Fluorescent RT-LAMP Mix 6μL 6μL 6μL each 20×Primer Mix 1μL 1μL 1μL each Template-primer Mix 5μL - - N+2 samples - - 1-13μL each ddH2O 8μL 13μL Add to 20μL 1. Seal reaction vessels. 2. Incubate at 65°C for 60 minutes in a real-time PCR machine. Collect signal in the SYBR channel in every 60 seconds. 3. The two positive reactions should have S-shaped fluorescent signal. The two negative controls should have no S-shaped fluorescent signal. 4. If there is amplification in NC or there is no amplification in PC, the whole experiment is invalid. There is no need to analyze the results of the N+2 samples. Refer to Troubleshooting section. 5. If there is no amplification in NC or there is amplification in PC, the whole experiment is valid. Analyze and record the results of the N+2 samples. The results from fluorescent detection can be downloaded from the real-time PCR machine. Troubleshooting 1. There is amplification in NC. 1. This result suggests possible carryover contamination in working environment and/or reagents. To avoid carryover contamination, clean working areas and equipment using a 10% chlorine bleach solution, use new (un-contaminated) reagents, primer stocks, water, etc., use UNG-RT-LAMP Kit, perform experiments in a new (un-contaminated) room. 2. This result also suggests nonspecific amplification. To avoid nonspecific amplification, do amplification at temperature of 65°C -70°C，or put the reaction tubes to 65°C as soon as the reaction is set，or use a new set of primer for the targeted DNA region. 2. There is no amplification in PC 1. This result suggests failures in machine or reagents，use new machine or new set of reagents. Limited Product Warranty Biofargo，Inc. warrants its products as set forth in the Biofargo’s General Terms and Conditions of Sale at www.biofargousa.com. If you have any questions, please contact Biofargo @ www.biofargo.com. The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, BIOFARGO INC. WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
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