Do you have a methylene blue dot blot staining protocol?

We have found that the methylene blue staining of the amounts of DNA spotted when performing a DNA dot blot can be tricky and may need optimization.

1. It is very important to perform the methylene blue staining prior to blocking (Reference Step C in the DNA dot blot protocol).
2. Stain the membrane with Methylene Blue Stain stain for 10 min at room temperature while gently shaking. Reagents we have had success with in our own internal testing include Methylene Blue Stain (MB 119, Molecular Research Center) and 1% Methylene Blue solution (M9140, Sigma, diluted in water).
3. Wash the membrane, 2-3 times with dH20 (2-5 min each) until desired results are obtained.
4. Once desired results are obtained, image blot immediately as the stain tends to fade.
5. After imaging, proceed with blocking and antibody incubation with the membrane (step C). Remaining methylene blue should not affect antibody binding.

Last updated: September 12, 2024