New England Biolabs, L4001P, LyoPrime Luna® Probe One-Step RT-qPCR Mix with UDG

Supplied as a lyophilized cake, the LyoPrime Luna Related Categories Luna® qPCR & RT-qPCR Applications Probe-based qPCR & RT-qPCR,, qPCR & RT-qPCR,, Two-Step RT-qPCR, FAQ Q: How should the LyoPrime Luna mix be stored? A: Vial format: Prior to use, LyoPrime Luna products can be stored at room temperature (15 to 25°C) and protected from light (e.g. in the original product box or another closed box). The glass vial, cap and rubber stopper are impermeable to moisture, allowing storage without desiccant. Storage at -20°C is neither necessary nor recommended prior to rehydration. Additional storage and rehydration information can be found here. Plate format: Prior to use, the LyoPrime Luna Mix should be stored at room temperature (15°C to 25°C) unopened in its original foil pouch. The foil pouch and desiccant inside protect the lyophilized reagent from atmospheric moisture and light. Any unused strip tubes should immediately be placed back in the foil pouch with the desiccant, sealing the zip-lock well and storing at room temperature. Additional storage and rehydration information can be found here. Q: How do I rehydrate the LyoPrime Luna mix? Can it be resuspended at a higher concentration? A: Vial format: To rehydrate the lyophilized mix as a 2X Master Mix, remove the vial cap and stopper, add 675 μl nuclease-free water, and briefly mix by pipetting or gentle vortexing. The lyophilized mix can be rehydrated at up to 4X to accommodate higher sample volumes. A video demonstration can be found here. Please also see the protocol page for additional information. Plate format: The lyophilized mix in each well can be rehydrated directly with primers, probes and template to achieve 1X Master Mix (20 μl reaction volume). Therefore, it is highly recommended to prepare an assay mix including primer and probe(s) that is dispensed into each well. Following adding assay mix, briefly mix by pipetting or vortexing using microplate shaker and then add template. Rehydration information can be found on the protocol page here and is also demonstrated in this video. Q: Are reaction conditions the same for lyophilized and liquid Luna mixes? A: Yes, reaction conditions for the LyoPrime Luna® Probe One-Step RT-qPCR Mix with UDG (NEB #L4001) are the same as for our corresponding liquid product, the Luna Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019). Both mixes include the engineered Luna WarmStart Reverse Transcriptase, which possesses higher thermostability than many other RTs and has an optimal reaction temperature of 55°C. Please see the protocol page (L4001 vial or L4001 plate) for additional details on reaction setup and cycling conditions. Q: When I dissolve the lyophilized LyoPrime Luna®  Probe product, the solution at first appears turbid. Is this normal? A: Yes, this is normal. The lyophilized cake in vial format dissolves immediately, but the release of air from the cake results in a temporary increase turbidity. The air will naturally degas from solution over ~20 seconds or after gentle vortexing. In the 96-well plate-based format, gentle mixing or vortexing results in immediate dissolution of the cake. Q: Can I set up my Luna® RT-qPCR at room temperature? A: Yes. Luna RT-qPCR can be set up at room temperature, but for best results, reactions should be kept on ice prior to thermocycling. Both enzymes present in the kit, Hot Start Taq DNA Polymerase and WarmStart® Luna Reverse Transcriptase, are controlled by temperature-sensitive reversible inhibitors (SOMAmer®). These SOMAmers inhibit polymerase activity below 45°C, preventing undesired nonspecific activity until the reaction is heated above the release temperature during the reverse transcription step. Q: Why is the Luna® qPCR Mix blue? Will this dye interfere with detection? A: The Luna qPCR Mix contains an inert visual reference dye that gives the mix a clear blue color. This colored appearance makes it easier to identify which wells of a qPCR plate have been loaded with reagents, which can be difficult with the small well size and opaque nature of many 96- and 384-well qPCR plates. The blue dye has been extensively evaluated in PCR and qPCR and it does not inhibit or interfere with qPCR detection. Q: Can I run the Luna® qPCR Mix on my qPCR instrument? A: The following Luna qPCR mixes contain a universal passive reference dye that enables compatibility with a wide variety of real-time instruments, including those that use no passive reference normalization dye (e.g. Bio-Rad® CFX), a low concentration passive reference (e.g. Applied Biosystems® 7500 or QuantStudio®), or a high concentration passive reference (e.g. Applied Biosystems 7900 or StepOne®). Luna Universal qPCR Master Mix (NEB #M3003) Luna Universal Probe qPCR Master Mix (NEB #M3004) Luna Universal One-Step RT-qPCR Kit (NEB #E3005) Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006) Luna Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019) LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG (NEB #L4001) The Luna Probe One-Step RT-qPCR Kit (No ROX) (NEB #E3007) and Luna Probe One-Step RT-qPCR 4X Mix with UDG (No ROX) (NEB #M3029) lack a reference dye and can be used with any instrument that does not require ROX normalization. Please refer to instrument manufacturer’s instructions for greater details. Additionally, the Luna Mix can be used with standard or fast cycling conditions. Q: Do I need to add ROX? A: Some real-time instruments recommend the use of a passive reference dye (typically ROX) to overcome well-to-well variations that could be caused by machine limitations such as “edge effect”, bubbles, small differences in volume, and autofluorescence from dust or particulates in the reaction. However, ROX normalization does little to the variations caused by pipetting errors of templates/primers, heterogeneous mixing, and evaporation/condensation issues. The following Luna® qPCR products contain a universal passive reference dye that enables compatibility with a variety of real-time instruments, including those that use no passive reference normalization and those that use a low or high amount of passive reference dye (ROX). Luna Universal qPCR Master Mix (NEB #M3003) Luna Universal Probe qPCR Master Mix (NEB #M3004) Luna Universal One-Step RT-qPCR Kit (NEB #E3005) Luna Universal Probe One-Step RT-qPCR Kit (NEB #E3006) Luna Probe One-Step RT-qPCR 4X Mix with UDG (NEB #M3019) LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG (NEB #L4001) The Luna Probe One-Step RT-qPCR Kit (No ROX) (NEB #E3007) ) and Luna Probe One-Step RT-qPCR 4X Mix with UDG (No ROX) (NEB #M3029) lack a reference dye and can be used with any instrument that does not require ROX normalization. Please refer to instrument manufacturer’s instructions for greater details. qPCR Instrument ROX Bio-Rad® iQ™5, CFX96, CFX384, Opticon Roche Lightcycler® Qiagen Rotor-Gene™ Eppendorf Mastercycler® Cepheid® SmartCycler® Not recommended/required Applied Biosystems® 7500, 7500 Fast, QuantStudio™, ViiA7™ Agilent Mx™ Low ROX (~50 nM final) Applied Biosystems® 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™ High ROX (~500 nM final) Q: What RNA samples can be used in RT-qPCR with the Luna® Mix? A: For best results, we recommend an extraction or purification of the RNA starting material. The Luna RT-qPCR Kit works with nucleic acids from a variety of sample types purified using typical column-based methods (e.g. Monarch® RNA Miniprep Kit (NEB #T2010)). When performing studies using mammalian cell cultures, cell lysates generated by Luna Cell Ready Lysis Module (NEB #E3032) can be added to all Luna one-step RT-qPCR reagents. In addition, numerous studies have directly demonstrated the compatibility of Luna reagents with saliva and other clinically relevant samples using crude sample prep protocols. SalivaDirect is one example. Q: How much RNA template should I use in my RT-qPCR reaction? A: The Luna® Universal One-Step RT-qPCR Kit works well with purified RNA templates including total RNA, mRNA, and in vitro transcript samples. Amount in 20 μl reaction total RNA ≤ 1 μg poly(A)-RNA ≤ 0.1 μg in vitro transcripts ≤ 10⌃9 copies Q: Can I run multiplex reactions with the Luna® Probe One-Step RT-qPCR Kits? Do I need to change my reaction conditions? A: Yes, multiplex reactions amplifying up to 5 targets have been successfully run using the Luna Universal Probe RT-qPCR Kits. Select the different fluorophores corresponding to separate detection channels of the real-time instrument. Each target should be tested in a singleplex reaction first. For each amplicon, begin by including 400 nM of forward and reverse primer and 200 nM probe in each reaction. If performance of a particular amplicon is seen to deteriorate when used in a multiplex assay as compared to its singleplex results, reactions may require optimization. It can be helpful to reduce the primer concentrations for an abundant or high-copy target and/or increase the primer concentrations for the low abundance or low-copy targets. Further optimization may be needed as multiplexity increases, using the following parameters as guidelines: primer 100–900 nM, probe 100–500 nM. Example of a 3-plex reaction using the Luna Universal One-Step RT-qPCR Probe Kit on an Applied Biosystems™ 7500 Fast instrument: Q: Can I use a ROX-labeled probe with the Luna® Probe Mixes that contain a universal ROX reference dye? A: Yes. However, the passive reference normalization requirements of the real-time instrument should be considered. For instruments where no ROX normalization is used, a ROX probe can generally be used without concern or instrument modification. For instruments where ROX is used for normalization, ROX probes are typically not recommended and this option is not available in the typical instrument settings. It is still possible to use ROX probes provided that ROX normalization is turned off, but the benefits of the normalization will be lost. For those preferring a ROX-free mix, please see the Luna Probe One-Step RT-qPCR Kit (No ROX) (NEB# E3007) or the Luna Probe One-Step RT-qPCR 4X Mix with UDG (No ROX) (NEB #M3029), or contact info@neb.com. Q: Other companies recommend a 2-min incubation at 25°C for carryover prevention, is a 30-sec incubation at 25°C sufficient? A: Yes, given the robust activity of NEB’s thermolabile UDG, we see the degradation of up to approximately 100,000 copies of mock contamination input using a 30-sec incubation step at 25°C. Since carryover contamination typically involves much lower input amounts, there is no need to extend the incubation. Q: Can I use the Luna Probe RT-qPCR mixes for viral detection? A: Yes, the Luna Probe RT-qPCR Mixes are particularly well-suited to enable sensitive detection of viral targets, including detection of the SARS-CoV-2 virus. Examples of how people have used Luna and other products can be found on our COVID-19 research page. Q: What are the differences between the Luna One-Step RT-qPCR products (NEB #E3006, #E3007, #M3019, #M3029 and #L4001)? A: These Luna products are all optimized for one-step RT-qPCR by probe detection. NEB #M3019/#M0329 are one-tube 4X master mixes The NEB #E3006/#E3007 products contain separate tubes for the 2X reaction mix and 20X enzyme mix. Also, the M3019 product includes not only dUTP, but also a thermolabile UDG, to fully enable carryover prevention. NEB #L4001 is a lyophilized version of NEB#M3019 and is available in vials or 96-well plates. NEB #E3007 and #M3029 lack a universal ROX passive reference dye. Available mixes differ with regards to format, storage temperature, and inclusion of thermolabile UDG for carryover prevention, as outlined in the table below. NEB #M3019 Luna Probe One-Step RT-qPCR 4X Mix with UDG NEB #M3029 Luna Probe One-Step RT-qPCR 4X Mix with UDG (No ROX) NEB #E3006 Luna Universal Probe One-Step RT-qPCR Kit NEB #E3007 Luna Probe One-Step RT-qPCR Kit (No ROX) NEB #L4001 LyoPrime Luna Probe One-Step RT-qPCR Mix with UDG Format 4X Master Mix 4X Master Mix 2X Reaction Mix 20X Enzyme Mix 2X Reaction Mix 20X Enzyme Mix Lyophilized Master Mix; rehydrate at 2-4X Carryover prevention included Y (dUTP and thermolabile UDG included) Y {dUTP and thermolabile UDG included) N (dUTP included; requires UDG addition) N (dUTP included; requires UDG addition) Y (dUTP and thermolabile UDG included) Normalization dye (universal ROX) Y N Y N Y Multiplexing Y (up to 5-plex) Y (up to 5-plex) Y (up to 5-plex) Y (up to 5-plex) Y (up to 5-plex) Maximum sample volume (20 µl RT-qPCR reaction) up to 10 µl up to 10 µl up to 8 µl up to 8 µl up to 10 µl Standard cycling condition 25°C for 30 sec. (UDG treatment); 55°C for 10 min; 95°C for 1 min; 45 cycles of 95°C for 10 sec., 60°C for 30 sec. 25°C for 30 sec. (UDG treatment); 55°C for 10 min; 95°C for 1 min; 45 cycles of 95°C for 10 sec., 60°C for 30 sec. 55°C for 10 min; 95°C for 1 min; 45 cycles of 95°C for 10 sec., 60°C for 30 sec. 55°C for 10 min; 95°C for 1 min; 45 cycles of 95°C for 10 sec., 60°C for 30 sec. 25°C for 30 sec. (UDG treatment); 55°C for 10 min; 95°C for 1 min; 45 cycles of 95°C for 10 sec., 60°C for 30 sec. Storage temperature -20°C -20°C -20°C -20°C Room temperature (15°C to 25°C) Q: For L4001P - Do I need to use the entire Luna plate in a single experiment? How are the 8-well strip tubes separated? A: The 96 well plate and optical lids are provided in a tear-off format allowing use of the entire plate directly or partially. The twelve 8-well strips are partially attached to each other, so the required number of 8-well strip tubes can be separated by gently tearing away the desired number of strip tubes. The 8-well strips are clicked in the yellow shell frame and the yellow shell frame adaptor must be removed to separate one or more strip tubes. The 8-well strips and lids are labeled 1 to 12 at the bottom to provide a tracking system independent of the yellow shell frame adaptor. Please check Plate Information and Instrument Compatibility section in the protocol for more information. Q: For L4001P - How do I store unused 8-well strip tubes after opening the foil pouch? How long can I keep unused 8-well strip tubes? A: Any unused 8-well strip tubes should be immediately resealed in the original zip-lock foil pouch with the provided desiccant and can be stored at room temperature. It is recommended to use these tubes within 1 week of opening of the foil pouch for the best results. Q: For L4001P - How long can I keep the plate or the 8-well strip tubes out of the foil pouch before setting up the reaction? A: After taking the plate out of the foil pouch, it is recommended to hydrate the lyophilized reagent intended for use within 1 hour. Q: For L4001P -  What is the purpose of yellow shell adaptor? A: The strip tubes are provided in a single use yellow shell frame semi-skirted adaptor. This yellow adaptor has chamfered edge at the upper left corner (A1), which supports the use of the product on ABI /Life Technologies® FAST Cyclers and robotic systems. The yellow shell frame provides rigidity to the plate, so it is highly recommended to use for full plate runs in the ABI /Life Technologies FAST Cyclers. If the plate will be used partially, individual 8-well strips can be used with or without the yellow shell frame in ABI /Life Technologies FAST Cyclers. Use of other instrument platforms (e.g., Bio-Rad®) will require removal of the yellow shell adaptor prior to thermocycling. Please see the Plate Information and Instrument Compatibility section in the protocol for recommendations on the use of the yellow shell adaptor in common real-time PCR instruments. Q: For L4001P - Can I reuse the yellow shell adaptor? A: The yellow shell adaptor is intended for single-use to prevent cross contamination between RT-qPCR runs. It should be discarded along with the strip tubes after the run is complete. Separating individual strips from the yellow shell adaptor post amplification may result in the unintended opening of the caps and potential amplicon contamination events. If additional yellow shell frame adaptors are needed, they can be purchased from BIOplastics (product code AB19805G.) Q: For L4001P - Why is there a cardboard tray in the package? A: The cardboard tray is included in the packaging to support the plate during shipping. It can be recycled upon opening the package, or it can be used as a support for the plate during room temperature setup.