I need to quantify synthetic oligonucleotides, so I'd like to synthesize the shortest sequence possible. I was wondering whether I need a spacer between the primer and the probe complementary region on the template. Thank you very much.
When your primer and probe are on the same strand you need at least 5 bases (I always leave around 10), for the primer on the other strand you don't need to leave any space (I prefer to leave 1-2 bases anyway).
I would recommend you to use LNA (locked nucleic acid) probes, you can shorten your probe considerably (they are more expensive).
Hi,
It would be better if you keep at least 4-5 bp distance between one of your primers and probe. but longer distance will not make severe problem in your result.
When your primer and probe are on the same strand you need at least 5 bases (I always leave around 10), for the primer on the other strand you don't need to leave any space (I prefer to leave 1-2 bases anyway).
I would recommend you to use LNA (locked nucleic acid) probes, you can shorten your probe considerably (they are more expensive).
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