WarmStart LAMP Kit (DNA & RNA) Protocol (E1700)
A 10X LAMP Primer Mix contains:
| PRIMER |
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. Prepare reaction mix as described below. Volumes are listed for a 25 μl LAMP reaction, but other volumes (10, 20, 50 μl etc.) are all effective; if desired, adjust volumes accordingly. A 1 µl target DNA volume is shown; if higher sample volumes are needed, adjust volume of H2O. For non-template reactions add equivalent volume of H2O or sample storage buffer.
DNA TARGET DETECTION |
RNA TARGET DETECTION |
NO- TEMPLATE CONTROL (NTC) |
|
| WarmStart LAMP 2X Master Mix |
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 |
1 µl |
– | – |
| Target RNA |
– | 1 µl |
– |
| dH2O | 8.5 µl |
8.5 µl |
9.5 µl |
| Total Volume |
25 µl |
25 µl |
25 µl |
3. Vortex reaction mix and centrifuge to collect material.
4. Pipet 24 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 be 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.