FAQ: Can I use NaOH (sodium hydroxide) instead of formamide to denature my DNA prior to the deamination reaction step in the E5hmC-seq protocol?

Yes, it is possible to use NaOH, although formamide is preferred. If using NaOH it is critical that the concentration of the NaOH solution is accurate, and the volume added to the reaction is exactly 4 μl.

  • NaOH is highly corrosive. Local and institutional safety guidelines must be followed when working with NaOH.
  • If you are making a dilution from 2 N NaOH, ensure the stock solution has at least a pH of 12.5. Verify the pH of the stock solution prior to use.
  • Avoid inaccurate dilutions by making a large enough volume of diluted NaOH to minimize pipetting errors. We recommend making at least 1 ml. Prepare NaOH dilutions fresh or store diluted aliquots at 20°C for up to 6 months to avoid a change in concentration.
  • Solid NaOH is deliquescent (it absorbs water from the atmosphere). It cannot be weighed accurately. Therefore, solutions prepared from NaOH pellets must be titrated to achieve the proper concentration.