I am having problems with high efficiency of my primers and one reason could be the formation of primer dimers.
I dont see any big peak on the melt curve, but I dont know if this increased peak is a primer dimer as well. Also, I dont see any smaller band in my gel.
What should I do?
I have used serial cDNA dilutions from 800ng RNA, 400, 200, 100 and 50, but with this I used up all 20uL of my RT sample. If I dilute 10x to get at least 200uL of cDNA, then I would have to decrease even more the concentration of my primers, right?
Hi. you can run your real time Products in Agarose gel 1-2% and i think you can see primer dimer. you should use the 50bp ladder to distinguish. :)
Hi Simone,
as Ehsan said it's a good way to see primer dimers. an other way is bioinformatics and an in silico PCR (https://www.eurofinsgenomics.eu/en/dna-rna-oligonucleotides/oligo-design-more/primer-design-tools.aspx) could help you to see if primer dimers are possible.
fred
Use low concentration of primers to minimize/eliminate formation of primer dimers.
Design new primers, Your melting curve looks OKish, I doubt that you will completelly resolve the primer dimers issue just changing conditions (i.e. concentration or annealing T) . Best of luck! a
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