Product Description Biofargo Colorimetric RT-LAMP Kit is designed to provide a one-step solution for Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) of RNA and an equipment-free examination of the amplification results with naked eyes. It has the following features： Optimized RT-LAMP mix, contains colorimetric dyes that enables the examination of RT-LAMP results with naked eyes. 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#211209-50 is sufficient for 50 reactions of 20μL volume, and CAT#211209-500 for 500 reactions of 20μL volume. Input Sample Requirements Perform RNA 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 another negative control. Kit Contents Component CAT#:211209-50 CAT#:211209-500 Colorimetric 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 Paraffin oil, molecular biology grade 6 Heat block, water bath or 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. But if agarose gel electrophoresis or other post-reaction analysis is desired, perform the analysis in a separated room. 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 100uM stocks to make 100μL 20×Primer Mix according to the following table. If volumes other than 100uL (50μL, 200μL etc.) is desired, adjust volumes accordingly. Components to add Volume to add Final Concentration in 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 Colorimetric 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 Colorimetric 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 another for RT-LAMP Negative Control (NC) . PC NC N+2 Colorimetric 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 Add 30uL paraffin oil if heat block, water bath is used as incubator. If a PCR machine is used as incubator, turn on the heat lid. Seal reaction vessels. Incubate at 65°C for 60 minutes. Remove tubes or vessels from incubation and examine by eye. Positive reactions will have turned green while negative controls should turn light green. If color difference is not obvious, allow reaction to cool at room temperature. If there is amplification in NC or there is no amplification in PC，the whole experiment is invalid. There is no need to analysis the results of the N+2 samples. Refer to Troubleshooting section. If there is no amplification in NC or there is amplification in PC，the whole experiment is valid. Analyze and record he results of the N+2 samples. The result can be photographed or scanned to record the colorimetric results. Troubleshooting There is amplification in NC. 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. This result also suggests nonspecific amplification. To avoid nonspecific amplification, do amplification at temperature of 65°C-70°C，put the reaction tubes to 65°C as soon as the reaction is set，use a new set of primer for the targeted DNA region. There is no amplification in PC 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.tiandzusa.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.
