Nov 29, 2022
Colorimetric LAMP/RT-LAMP Protocol
- Felipe Navarro Martínez1,2,
- Fernan Federici1,2
- 1Laboratorio de Tecnologías Libres;
- 2Millennium Institute for Integrative Biology (iBio), Santiago, Chile
- Laboratorio de Tecnologias Libres
- Reclone.org (The Reagent Collaboration Network)
Protocol Citation: Felipe Navarro Martínez, Fernan Federici 2022. Colorimetric LAMP/RT-LAMP Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvor3x7v4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 10, 2022
Last Modified: November 29, 2022
Protocol Integer ID: 72588
Keywords: lamp reactions with homemade buffer, lamp reaction protocol, colorimetric lamp, lamp reaction, colorimetric method, lamp protocol, reaction ph, lamp protocol this protocol, ph indicator, inherent proton production by dna polymerase, buffered reaction, lamp, drop in the reaction ph, inherent proton production, ph, enzyme, dna polymerase, amplification process, phenol, brewed bstlf, homemade buffer, mmlv enzyme
Abstract
This protocol describes how to perform colorimetric LAMP / RT-LAMP reactions with homemade buffers, together with home-brewed BstLF and MMLV enzymes.
It is based on previous protocols of our lab (see LAMP/RT-LAMP Reaction protocol), but since two of the most common indicators used in colorimetric LAMP (phenol red and neutral red) require weakly buffered reactions, the buffer composition needed to be changed. This was done according to indications by Poole et al (2017).
The colorimetric methods rely on the inherent proton production by DNA polymerases during the amplification process, which leads to a drop in the reaction pH that can be followed using these pH indicators. The results can be seen directly with the "naked eye", without the need for equipment or hands-on processing (Tanner et al., 2015).
Color change of neutral red and phenol red indicator dyes in LAMP reactions.
Before amplification (T0), reactions containing neutral red are colorless. Samples turn pink if positive or a brownish yellow if negative as shown after a sixty-minute (T60) amplification. Reactions containing phenol red are pink at T0 and remain pink if negative but turn yellow if positive as shown here after a sixty-minute (T60) amplification. [Figure from Poole et al (2017)].
Troubleshooting
2X Buffer Preparation
Prepare 1000 µL of 2X Colorimetric Buffer Mix according to the following table:
- Two main differences compared to non-colorimetric LAMP reaction are the addition of dNTPs in the buffer, as they influence the reaction pH, and the removal of Tris-HCl as this component is vital to maintain the pH of the buffer at a stable point, which is not desirable for pH-dependent detection
Measure the reaction pH with strips and adjust pH using NaOH 250 mM to a final value of pH 8-9.
Note
The pH of the original solution should be around 5-6 before adjusting.
Add nuclease-free water to a final volume of 1000 µL . Make aliquots and store them at 4 °C until use.
Prepare the LAMP or RT-LAMP reaction mix On ice according to the following tables:
LAMP1 step
- Neutral red can be replaced with another pH indicator like phenol red
- To adjust the concentration of BstLF enzyme use its 1x Storage Buffer
- 2 uL of DNA could also be added to avoid the use (and possible contamination due to reopening) of nuclease-free water
- Reactions can also be scaled up to higher volumes, especially if you are working with a higher sample volume
Incubate the reactions at 65ºc for 35 minutes and observe results with the naked eye.
Expected result
These are expected results with the use of neutral red as a colorimetric indicator, with negative reactions (NTC) staying yellow and positive reactions (in this case, six5) turning pink: