MethyleneBlueDNAstainingprotocol

Methylene Blue DNA staining protocol

Protocol:

1. Load 2-5X the amount of DNA that would give bands of moderate intensity on an ethidium bromide stained gel. Typically this is something on the order of 0.5-2.5 µg of a 1 kb fragment on a 30 ml 1% mini gel. These numbers are guess-timates so your milage may vary.

2. Run the gel normally and then place in a 0.002% methylene blue (w/v, Sigma M-4159) solution in 0.1X TAE (0.004M Tris 0.0001 M EDTA) for 1-4 h at room temp (22°C) or overnight at 4°C. Diffusion of the DNA does not seem to be a problem for fragments as small as 100 bp (3% Nusieve:1%agarose gel). This avoids background issues associated with staining with 0.02% methylene blue for 30-60 min and then destaining for what seems to be forever

3. If destaining is needed to increase the visibility of the bands place the gel in 0.1X TAE with gentle agitation changing the buffer every 30 - 60 min until you are satisfied with the degree of destaining.

Notes:

This method primarily eliminates the damage of DNA by uv irradiation. DNA isolated from MB stained gels should transform frozen competant E. coli (XL1-Blue and DH5) cells on the order of 20-50 fold more efficiently than EB isolated DNA. Factors influencing improved efficiency are: time factor (degradation, etc.), transilluminator wavelength and intensity, and the %AT of your DNA to mention a few. One of the advantages of MB staining is the elimination of several of the variables.

Both FMC GTG agarose and Nusieve GTG perform very well. Synergel is incompatible with MB (very high background). MB should be compatible with polyacrylamide (even less of a background problem).

NuSieve:Agarose (3:1, 4% final) gels stain very nicely and dsDNA as small as 75 bp is easily visualized.