Use this category for questions related to various quantitative PCR methods, including real time PCR, reverse transcription (RT)/PCR, qPCR, etc. For general PCR methods, continue to use the DNA/General PCR forum
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Been running qPCR for several years but have come across a new issue over the past couple weeks. We recently designed about 15 new TaqMan assays that target various pathogens. Most of the assays work flawlessly but 3 - 4 of them are exhibiting a very strange phenomenon that I haven't seen previously. The amplification is beginning normally but then somewhere midway through the amplifcation, the signal is suddenly decreasing as shown in the attached document.
I've tried different purified DNA samples (and the same DNA has been tested successfully with other TaqMan sets), new primers, different master mixes, etc to no avail. We run dozens of other assays daily using the same reagents and they work fine but these particular assays always display this crashing effect to different degrees. I did reorder the probes to test and are waiting for them to arrive.
I've seen somehing like this before with hybridization probes (i.e. the 'hook' effect) but these are TaqMan probes so that seems unlikely. Has anybody seen this or have any thoughts on what might be happening?
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