Protocol for LyoPrime WarmStart^™ Fluorescent LAMP/RT-LAMP (with UDG), 96-well plate (NEB #L4401P)

Please read all instructions before opening the moisture barrier foil pouch for this product.

Introduction

Loop-Mediated Isothermal Amplification (LAMP) offers a robust, simple, one-step method to detect target nucleic acids (DNA and RNA). The LyoPrime WarmStart^TM Fluorescent LAMP/RT-LAMP Mix (with UDG) (NEB #L4401P) is provided lyophilized in a ‘ready-to-use’ 96-well plate format, and it is shipped and stored at room temperature before use.

All necessary components required for a 25 μL LAMP assay are provided in each well of the 96-well plate, except primers and template input (DNA or RNA). LAMP Fluorescent Dye (NEB #B1700) at 0.5X concentration is included in L4401 to enable detection of amplification via real-time fluorescence. Antarctic Thermolabile UDG and dUTP are also included to reduce the risk of carryover contamination between reactions.

This product is available as a single 96-well plate. It also can be customized to meet your specific application needs by contacting us.

Plate Information and Instrument CompatibilityThe LyoPrime WarmStart Fluorescent LAMP/RT-LAMP Mix (with UDG) is supplied in a low profile 96-well x 0.1 mL semi-skirted PCR plate, capped with robust optical lids in a tear-off format. The strip tubes are in a single-use yellow shell frame semi-skirted adaptor and sit in a cardboard insert. The 96-well plate is packaged in a foil pouch to protect the lyophilized reagent from atmospheric moisture. Please see the Storage Section for information on the stability of the plate both before and after the foil pouch has been opened. If desired, the cardboard insert can be removed and recycled upon opening the pouch. The product is compatible across several common instrument platforms including Bio-Rad^® CFX96 Touch/Opus, Applied Biosystems^® FAST Cyclers and Roche^® Lightcycler^®.

Plate usage: The twelve 8-well strip tubes are partially attached, allowing the use of the entire plate or partial consumption by separating one or more individual 8-well strip tubes after removal from the yellow shell frame adaptor. 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. The yellow shell frame adaptor must be removed to separate one or more strip tubes, but the tubes can be clicked back into the adaptor if necessary. If a single strip tube or partial plate is used in combination with the yellow shell frame grid (optional), we recommend disposal of the yellow shell frame adaptor along with the PCR tubes post-amplification to avoid amplicon contamination events that could occur with additional tube/adaptor manipulation. Additional yellow shell frame adaptors can be purchased from BIOplastics (product code AB19805G.)

 Instrument compatibility: The yellow adaptor has a 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 the adaptor for full plate runs in the ABI/Life Technologies FAST Cyclers. If the plate is used partially, individual 8-well strips can be used with or without the yellow shell frame. Note that removing tubes from the yellow shell frame adaptor may be recommended for thermocycling on some instrument platforms. Please refer to the table below for recommendations on using the yellow shell adaptor in common real-time PCR instruments.

Storage

Before use, store the LyoPrime LAMP Mix at room temperature (15°C to 25°C), unopened, in its original foil pouch. The product has a shelf life of 24 months when stored properly under these conditions. The foil pouch and desiccant inside protect the lyophilized reagent from atmospheric moisture. After opening the foil pouch, it is recommended to hydrate the lyophilized reagent intended for use within 1 hour. Any unused strip tubes should immediately be placed back in the foil pouch with the desiccant, sealed securely, and stored at room temperature.  Failure to do so will result in a gummy reagent that is difficult to rehydrate, and product performance may be impacted. It is recommended that all strip tubes be used within 1 week of opening the foil pouch for best results.

Rehydration

Remove the optical lids and add liquid components indicated in the protocol section below. It is highly recommended that an assay mix that includes primers be prepared before dispensing it into each well to eliminate volumetric errors and ensure proper rehydration of the LAMP mix. The minimum initial resuspension volume for the 25 μl reaction is 5 μl. Rehydrate the lyophilized materials by adding the assay mix to each well and mixing by pipetting up and down or gently vortexing using a microplate shaker before adding the template.

Reaction Setup

For simplicity in setting up reactions, we recommend making stocks of the LAMP primers at a usable concentration.

For example, we suggest a 10X Primer Mix containing all 6 LAMP primers.

A 10X LAMP Primer Mix contains:

Prepare primer stocks in nuclease-free water and store at –20°C for up to 2 years.

An example protocol for setting up a 25 μl reaction with the addition of 2 μl template input is given below.

1. Open the foil pouch by tearing along the pre-cut notch and removing the 96-well plate. If desired, remove and recycle the cardboard insert.
   
   
2. If using individual 8-well strip tubes OR for use on non-ABI instruments, remove the strip tubes from the yellow shell adaptor. To release the tubes from the frame grid, set the plate on a flat surface and press down firmly on the four corners of the frame grid. Then, carefully lift the tubes from the grid. Separate the desired number of 8-well strip tubes and place in a PCR rack. Immediately place any unused strip tubes back in the foil pouch with desiccant and seal securely. Click tubes back into the yellow shell adaptor if necessary. Please note that the yellow shell adaptor is not required for thermocycling on all instruments (see recommendations above).
   
   
3. Determine the total volume for the appropriate number of reactions, plus 10% overage, and prepare an assay mix of all components except template input, according to the table below.

    

   Component	Volume per 25 μl Reaction	Final Concentration
   LyoPrime WarmStartTM Fluorescent LAMP/RT-LAMP Mix (with UDG)	-	1X
   LAMP Primer Mix (10X)	2.5 μl	1X
   Template Input (DNA/ RNA)	2 μl	variable
   Nuclease-free Water	20.5 μl

4. Aliquot assay mix into the desired wells of the plate. For best results, ensure accurate and consistent pipetting volumes and minimize bubbles.
   
   
5. Mix the lyophilized pellets thoroughly but gently by pipetting up and down five times. Alternatively, vortex using a microplate shaker (30 seconds at ~1600 rpm). If necessary, collect liquid to the bottom of the tube by brief centrifugation.
   
   
6. Add DNA or RNA template to the plate. Cap the plate using the provided optical lids.
   
   
7. Spin tubes or plate briefly to remove bubbles and collect liquid (1 minute at 2,500–3,000 rpm).
   
   
8. Incubate tubes or plate at 65°C for 30 minutes in a real-time instrument for fluorescence detection and collect data in the SYBR^®/FAM channel of the instrument. Time can be extended as necessary for very low copy targets, challenging sample types, or reactions known to produce slower amplification times.
   
   
9. If reaction products will be manipulated or analyzed after LAMP is complete, Bst 2.0 and RTx can be inactivated by heating at > 80°C for 5 minutes. However, we typically recommend that assay tubes are NOT opened following incubation at 65°C and are disposed of immediately upon completion to avoid amplicon contamination.