This issue is occurring sporadically. I am wondering whether it may be a faulty machine part or my reagents/ cDNA. I do not think it is due to bad master mixes because I ran clean tests on the same day with the same master mixes. The disassociation curves are poor but my Ct values are all in an acceptable range (about 20-28). I also ran the amplicons on an agarose gel to confirm their presence and each was present.
Katie A Burnette
Just looking at the scale of the Y-axis in your second figure, you are getting very weak fluorescent signal. I'm guessing your dye is getting too old/too many freeze & thaw cycles. Remember that SYBR is very light and temperature sensitive. Try using a fresh batch of reagents and I think you'll fix the issue.
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