Hi everyone,
Recently I am working on oligonucleotide. I have a fluorophore tagged DNA for my experiment. My DNA is tagged with 6-FAM at 3' end. It is suggested that FAM tagged DNA should remain in solutions with 7.2 pH or higher to limit degradation. There are TE buffer or PBS which can be used to dissolve it. For my experiment I want to avoid EDTA and salt actually. But to maintain higher pH ,I have to go with a buffer solution. Now my question is:
Can I use phosphate buffer of pH 8 instead of TE buffer or PBS to dissolve this FAM tagged DNA?
If any other buffer solution will be useful, then please suggest me.
FAM, fluorescein derivatives are susceptible to a relatively high rate of photobleaching and are sensitive to acidic environments. The signal will rapidly degrade as the dye becomes protonated.
I will recommend dissolving the FAM-labeled DNA right before using it.
PBS buffer capacity is too weak at pH 8. You can use Tris or Tricine buffers.
You certainly can use the PBS buffer to dissolve oligos labeled with FAM. What is more important for the stability of the dye than the buffer is the amount of light (both from the sun and the electric lighting in your lab). All dyes including FAM bleach rather rapidly upon exposure to the light. The good practice is to keep the labeled oligos frozen and in the dark and have them on the bench only as long as it is needed for handling.
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