Hello everyone. I`m getting a good amplification in the LAMP reaction (see figures), but I have received no change in color of pH-sensitive dyes (f.c. 0.05-0.1 mM of the Phenol Red, Cresol Red, Phenolphthalein, Bromothymol Blue or Thymol Blue). I have tried to optimize the concentration of Bst DNA Polymerases (Large Fragment and 2.0, NEB), primers, dNTPs (f.c. 0.35-1.4 mM), isothermal buffers (pH 7.0-9.6), template (100 - 20 000 copies of the plasmid control), additional components (f.c. 0.8 M Betaine, 7.5% DMSO) and temperature (60-65 C), but results were similar. Does anyone know what can be my problem and how to solve it?
You would need to find a good buffer (no buffer, or less/weak buffer capacity) to make sure the system is sensitive to the PH changes.
I could try to test the buffer capacity by adding weak acid/basic (or buffer) to test whether the color changed with a small/tiny PH change.
Hi Junjie, I have tested 1x isothermal buffer with different pH values by dyes. All works fine in test wells (see photo).
Hi:
if the buffer is OK.
Could you run the gel to see whether the band is presented.
Or adjust the C. of Mg2+.
https://www.researchgate.net/post/Why_is_my_LAMP_reaction_not_turning_violet_to_sky-blue_after_adding_HNB
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