taq man probes non specific?
Posted 16 September 2010 - 11:52 AM
I have doing quite a few experiments examining expressing of a novel gene in a specific tissue type and I am getting postive readings from my taq man probes. I was just wondering what are the chances of the taq man probes being a false positive or more importantly has anybody ever experience getting a false positive with the taq man probes. I have also ran the resulting product on a gel and i get an expected band however i also get another unexpected band which is of a lesser intensity so that has just made me start to wonder.
Posted 17 September 2010 - 02:53 AM
If you have multiple products it means your primers are not specific or form primer-dimers (if the sequence is very small, like < 100bp, it's more likely it is a dimer). That doesn't mean the probe binds to that. So you only get fluorescent reads from specific sequence.
Anyway it's usually not so good to have unspecifities or dimers in your real-time reactions, you should increase the annealing temperature or redesign the primers.
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