Home Protocols WarmStart® Fluorescent LAMP/RT-LAMP Kit (with UDG) Protocols (NEB #E1708)

WarmStart® Fluorescent LAMP/RT-LAMP Kit (with UDG) Protocols (NEB #E1708)

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:

LAMP PRIMERS

10X CONCENTRATION (STOCK)

1X CONCENTRATION (FINAL)

FIP

16 µM

1.6 µM

BIP

16 µM

1.6 µM

F3

2 µM

0.2 µM

B3

2 µM

0.2 µM

Loop F

4 µM

0.4 µM

Loop B

4 µM

0.4 µM

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

  1. Thaw all components to be used at room temperature and place on ice. Vortex briefly to mix and centrifuge to collect material.

  2. An overview of the setup for one reaction is described in the table below. Volumes are listed for a 25 μl LAMP reaction, but other volumes (10, 20, 50 μl etc.) 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 volume of H2O (Y) to the desired final reaction volume. For no-template reactions, add equivalent volume of H2O or sample storage buffer. We recommend creating a reaction mix that lacks template (23 µl per reaction) such that the input sample can be added last. Room temperature set up of the reactions will enable carryover prevention.

    COMPONENT

    DNA TARGET
    DETECTION

    RNA TARGET
    DETECTION

    NO TEMPLATE CONTROL (NTC)

    WarmStart Multi-Purpose LAMP/RT-LAMP
    2X Master Mix (with UDG)

    12.5 µl

    12.5 µl

    12.5 µl

    Fluorescent dye (50X)*

    0.5 µl

    0.5 µl

    0.5 µl

    LAMP Primer Mix (10X)

    2.5 µl

    2.5 µl

    2.5 µl

    Target DNA

    2 µl

    –

    –

    Target RNA

    –

    2 µl

    –

    dH2O

    7.5 µl

    7.5 µl

    9.5 µl

    Total Volume

    25 µl

    25 µl

    25 µl

    * A 1X concentration of fluorescent dye is recommend for most real-time PCR instruments. Lower concentrations of dye (e.g., 0.1X) may be necessary on some instruments to avoid a saturated fluorescence signal.

  3. Vortex reaction mix and centrifuge to collect material.

  4. Pipet 23 μl per reaction into desired reaction vessels and add sample. Mix by vortexing and centrifuge to collect, or by pipetting if using a plate or other vessel.

  5. Seal reaction vessel.

  6. Incubate at 65°C for 30 minutes. Time can be extended as necessary for very low copy targets, challenging sample types, or reactions known to produce slower amplification times.

  7. 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.