I am finding a problem with the same primer which was yielding amplification. Now getting amplification but of different size and sometimes not getting any amplification for an already tested DNA sample.
https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cpmo.65
I used to make a 100 mM stock in 10 mM Tris-HCl pH 8 + 1 mM EDTA and keep it frozen at -20 °C. The stock can be frozen and thawed many times (> 10) and kept for many months, taking a few µl and making a fresh dilution in water each time to prepare the PCR mix. The important thing is to keep the stock in slightly alkaline conditions; EDTA inactivates nucleases and its residual concentration becomes negligible once the primer is diluted to concentrations required for PCR
It is generally not a good idea to thaw any biological material too many times but oligos are short and quite stable to freeze thaw cycles. The excellent IDT information attached measured activity at 30 freeze thaw cycles and found no difference in activity in pcr. I think that your problem could be caused by diluting the oligos in water not TE when the oligo gets depurinated by the acidity of water as time passes. Most lab purified water is pH about 5 and over time will absorb CO2 and become acidic. TE also stops nucleases from degrading the oligo. The best thing to do with your next batch of oligo is to make aliquots in TE then use one concentrated aliquot to make up your working dilution every couple of months so each aliquot only gets thawed/used once
I used to use my primer about 50 times at least (always -20 to RT and back). No problem at all. But that was for ordinary PCR. For qPCR I would be much more paranoid. And anyway, now l know that you shouldn't touch your 100 mM-stock too often. Make 10 mM aliquots from time to time and use those. Like this, your 100 mM-stock will stay fresh for years.
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