Protocol for LyoPrime WarmStart^™ Fluorescent LAMP/RT-LAMP Mix (with UDG) (NEB #L4401)

Introduction

Loop-Mediated Isothermal Amplification (LAMP) offers a robust, simple, and one-step method for detecting target nucleic acids (DNA and RNA). The LyoPrime WarmStart^TM Fluorescent LAMP/RT-LAMP Mix (with UDG) (NEB #L4401) is supplied in a lyophilized format, allowing it to be shipped and stored at room temperature before use. The LyoPrime LAMP mix is reconstituted simply by adding water. The mix contains all the necessary components for LAMP/RT-LAMP reactions except primers and sample 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.

Storage

Before use, the LyoPrime LAMP Mix should be stored at room temperature (15°C to 25°C) and protected from light. The product has a shelf life of 2 years when stored properly under these conditions. The glass vial, cap and rubber stopper are impermeable to moisture, allowing storage without desiccant. After the green cap and gray stopper are removed, the product should be rehydrated within 1 hour (see instructions below) and the gray stopper should be discarded.  After rehydration, the unused master mix can be stored protected from light at -20°C for up to 3 months or at 4°C for up to 3 weeks, using the green cap to seal the glass vial.

Rehydration

For use, remove the vial cap and stopper, add 675 μl nuclease-free water, and briefly mix by pipetting or gentle vortexing to reconstitute the lyophilized reagent cake as a 2X Fluorescent LAMP/RT-LAMP Master Mix. (To accommodate larger sample volumes, the lyophilized mix can be reconstituted as a 4X Master Mix by adding 337.5 μl nuclease-free water. However, reconstitution at higher concentrations than 2X may negatively impact time to detection for some targets).

Rehydration is rapid, and the lyophilized cake will typically dissolve within 1-2 seconds after water is added. Note that air from the cake will naturally degas from the solution over ~20 seconds or after gentle vortexing.

After rehydration, the LAMP/RT-LAMP master mix should be stored without the gray stopper. Seal the glass vial with the green cap for short-term storage at -20°C or 4°C.

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.

If using previously hydrated material, thaw completely and then briefly mix by gentle vortexing.

1. Rehydrate LyoPrime LAMP Mix as described above.
   
   
2. Thaw all components to be used at room temperature. Briefly mix other components by inversion, pipetting or gentle vortexing.
   
   
3. An overview of the setup for one reaction is described in the table below using a 2X LAMP/RT-LAMP Master Mix. Volumes are listed for a 25 μl LAMP reaction, but other volumes (10, 20, 50 µl,) are all possible; if desired, adjust volumes accordingly. A 2 µl sample input (DNA or RNA) volume is shown; if higher sample volumes are needed, adjust the volume of H₂O to the desired final reaction volume. For no-template reactions, add an equivalent volume of H₂O or sample storage buffer. We recommend creating an assay mix that lacks template (23 µl per reaction) such that the input sample can be added last. Room temperature setup of the reactions will enable carryover prevention.
   
   

   COMPONENT	DNA TARGET DETECTION	RNA TARGET DETECTION	NO TEMPLATE CONTROL (NTC)
   LyoPrime WarmStartTM Fluorescent LAMP/RT-LAMP Mix (with UDG) [rehydrated at 2X*]	12.5 µl	12.5 µl	12.5 µl
   LAMP Primer Mix (10X)	2.5 µl	2.5 µl	2.5 µl
   Target DNA	2 µl	–	–
   Target RNA	–	2 µl	–
   dH2O	8 µl	8 µl	10 µl
   Total Volume	25 µl	25 µl	25 µl

    *If the lyophilized mix is rehydrated to 4X, add 6.25 µl of 4X mix per 25 µl reaction and adjust the volume of H₂O accordingly.

    

4. Mix the LAMP assay mix thoroughly but gently by pipetting or vortexing. Collect liquid to the bottom of the tube by brief centrifugation.
   
   
5. Aliquot 23 µl of the LAMP assay mix into reaction tubes or plates at room temperature. Pipette 2 µl input (target DNA or RNA) into the reaction. Mix by vortexing and centrifuge to collect liquid, or by pipetting if using a plate or other reaction vessel.
   
   
6. Seal reactions with optically transparent caps or film.
   
   
7. Spin tubes or plates briefly to remove bubbles and collect liquid (1 minute at 2,500 - 3,000 rpm).
   
   
8. Incubate at 65°C for 30 minutes in a real-time instrument for fluorescent 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.