Hello Ritam Guha

The key difference between disodium EDTA dihydrate and tetrasodium EDTA dihydrate is the pH value of each compound. The pH of disodium EDTA dihydrate ranges between 4 and 6, not exceeding pH 7 while the pH of tetrasodium EDTA dihydrate ranges between 10 to 11.

Under optimum condition, for identifying agents with genotoxic activity, the alkaline (pH 13) version of the assay is suitable. The comet assay under (pH 13) version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA protein cross-linking, and SSB associated with incomplete excision repair sites.

Since you are going to perform the comet assay for the first time, the steps involved in the assay are:

1. Preparing slides with agarose gels,

2. Lysing cells to liberate DNA,

3. Exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB,

4. Electrophoresing the DNA using (pH 13) alkaline condition,

5. Alkali neutralization,

6. DNA staining,

7. Comet visualization, and data collection.

So, you will have to use tetrasodium EDTA dihydrate in comet assay for maintaining alkaline condition during the assay.

Best.

Malcolm Nobre

Thank you sir for your brief description and important tips.

Yes, you can you will merely have to adjust the mass specified for EDTA dihydrate by a factor of 1.12, and adjust the pH of the solution to the desired value with HCl, (the only net difference will be the generation of a small quantity of sodium chloride as a consequence of the pH adjustment which will be of no significance).