New England Biolabs, E1555L, LunaScript® Multiplex One-Step RT-PCR Kit

Related Categories RT-PCR,, cDNA Synthesis & Reverse Transcriptases Applications RT-PCR,, cDNA Synthesis,, RT-PCR & cDNA Synthesis FAQ Q: Can I set up my LunaScript Multiplex One-Step RT-PCR reactions at room temperature? A: Yes. The LunaScript Multiplex One-Step RT-PCR Enzyme Mix contains Luna WarmStart Reverse Transcriptase and Q5 Hot Start High Fidelity DNA Polymerase. This dual temperature-control of enzyme activities enables reaction setup and ensures stability of preassembled reactions at room temperature. Q: Can I add random hexamers or oligo (dT) primers to LunaScript Multiplex One-Step RT-PCR reactions? A: Using random hexamers or oligo (dT) primers with LunaScript One-Step RT-PCR reactions is not recommended. Inclusion of these primers may reduce the specificity of target amplification. Additionally, random hexamers require room temperature incubation for primer annealing, therefore, it is not compatible with Luna WarmStart Reverse Transcriptase, which requires full WarmStart activation at 50°C or higher. Instead, we recommend the use of gene-specific primers. Q: Can I reduce the reaction volume in the LunaScript Multiplex One-Step RT-PCR reactions? A: Yes, for many targets, the reaction volume can be cut in half (from the standard 25 µl to 12.5 µl) without compromising the PCR yield. Simply scale down the reagent volume while using the same amount of RNA input. Further reducing the volume may pose a challenge for manual setup due to pipetting difficulties. Q: How can I determine the annealing temperature for my LunaScript Multiplex One-Step RT-PCR reactions? A: Use of the NEB Tm Calculator is highly recommended for LunaScript Multiplex One-Step RT-PCR reactions as optimal annealing temperatures tend to be higher for Q5 Hot Start DNA Polymerase. When performing multiplex RT-PCR, use the annealing temperature calculated for the amplicon with the lowest annealing temperature. Q: Can I increase the reverse transcription time beyond the 10 minutes recommended in the LunaScript Multiplex One-Step RT-PCR protocol? A: The recommended RT time of 10 minutes at 55°C in the LunaScript Multiplex One-Step RT-PCR protocol is sufficient for most cDNA synthesis needs. Increasing the RT time to up to 20 minutes may increase product yield when the final primer concentration of each primer is less than 0.1 µM. Q: Are the PCR products generated with the LunaScript Multiplex One-Step RT-PCR Kit compatible with a TA cloning system? A: The PCR products created by Q5 Hot Start High-Fidelity DNA Polymerase contain blunt DNA ends and can be directly cloned using the NEB PCR Cloning Kit (NEB #E1202). A dA-tailing step is required for a TA-based cloning system. Q: What is the lowest RNA input amount recommended for the LunaScript Multiplex One-Step RT-PCR Kit? A: Successful target amplification depends on the quality of RNA samples and the abundance of the target gene expression. For example, a 570 bp GAPDH amplicon was detected from as low as 0.01 pg of high-quality human total RNA. Successful NGS libraries were also generated from as few as 10 copies of SARS-CoV-2 synthetic RNA using the ARTIC protocol. Q: What can I do if a particular amplicon failed in a multiplex reaction using the LunaScript Multiplex One-Step RT-PCR Kit? A: First, ensure that the amplicon length is within the recommended range of 100-1500 bp. If it is but fails to amplify in a multiplex reaction, perform a single-plex RT-PCR to evaluate the performance of the primers. Redesign the primers following guidelines in the General Tips and Consideration Section of the manual if no successful amplification was obtained in a single-plex RT-PCR. If the primer set works well in a single-plex RT-PCR, increase its concentration in the primer pool, (where each primer normally has the same concentration), by titration. This is likely to improve its amplification in a multiplex RT-PCR reaction. In addition to individual primer concentration rebalancing, as the number of targets increases in multiplex RT-PCR, the individual primer concentration may decrease, therefore, increasing the annealing time may also improve overall RT-PCR performance. For best results, we recommend empirically determining the annealing time in the context of primer concentration with the final total primer concentration between 1-4 µM. Q: Can I use the LunaScript Multiplex One-Step RT-PCR Kit to amplify long amplicons? A: The LunaScript Multiplex One-Step RT-PCR Kit is optimized for multiplex RT-PCR targeting amplicons between 100 – 1500 bp and can routinely amplify amplicons up to 3 kb in single-plex reactions. For long amplicons, please choose the OneTaq® One-Step RT-PCR kit (NEB #E5315) or two-step RT-PCR. For two-step RT-PCR, use the cDNA Synthesis Selection Chart to identify the RT and DNA Polymerase best suited for your application. Q: Can the LunaScript Multiplex One-Step RT-PCR Kit be used in the ARTIC workflow for RNA viral genome sequencing? A: Yes, this kit can be used to amplify RNA viral genomes with the multiplexing RT-PCR scheme used in the ARTIC workflow. Further optimization may be needed to achieve even amplicon balance and genome coverage. Q: How do I choose between One-Step RT-PCR and Two-Step RT-PCR protocols? A: Both one-step and two-step RT-PCR protocols offer robust detection of RNA targets. Depending on your experimental design and goals, you may prefer one protocol over the other. Please refer to the following table: Q: How do I choose between LunaScript Multiplex One-Step RT-PCR Kit and OneTaq One-Step RT-PCR Kit (NEB #E5315)? A: Both kits enable one-step RT-PCR. Please refer to the table below as a guideline to choosing between the LunaScript kit and the OneTaq One-Step RT-PCR Kit (NEB #E5315).