Hey Molecular Biology folks, I've got a question for you.
I'm developing TaqMan assays on cell lysates. I have a bunch of probes that when I run them for 40 cycles in RT-qPCR I get nice signal amplification and when I run the product on a 2% agarose gel after the qPCR i get a nice single amplicon (see the attached image). However, I have a few problem assays that also show strong signal amplification in the qPCR but when I run on a gel give the expected amplicon but also an additional higher weight product as well. My question is do these higher molecular weight bands matter? Clearly there may be some non specificity with my primers but my exon junction-spanning probes are what is giving signal and that should only be on the amplicon of interest. Can I ignore these other bands that are not the intended product? Interested to know how you all would handle this.
Hi Mike!
Sometimes I do the same - gel electrophoresis (2% agarose, non-denaturing conditions), especially in the case of hand made oligo design. Of course, I saw the results with some bands, but in all of case I saw the same bands. For example 100 b.p. target sequence (expected) and 250 b.p. unspecific. In any case I am doing Sanger sequencing to better understanding of reasons.
If I saw simple unspecific dimerization or other non specific sequence - it is ok, but if I see specific but longer sequence I shoul verify pseudogenes, may be rare allels etc. It could affect your results if you catch pseudogens or the simmilar gneetic sequence from different species in tour DNA pull.
As I can see (bright bands) you receive wide range of different products. Is that TaqMan or SYBR? DO you have sequencing results, melting curve?
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