I've optimised the PCR protocol for an unlabelled primer pair that target a range of pathogenic bacteria. The primers produce a amplicon of the predicted length at an optimum annealing temperature of 51 degrees C. As I plan to use T-RFLP, I've ordered the same primer pair with 6FAM and HEX labels attached to the 5' ends of the each primer respectively. However under the same PCR protocol, no amplicon is produced. Do I need to re-optimise the PCR annealing temperatures?
Fluorophores do affect the melting parameters of oligos although these effects strongly depend on the nucleotide context and are not easy to predict.
Analysis of melting curve of duplex between 5' labeled primer and complementary oligo with appropriate 3' quencher in PCR conditions can show you the annealing temperature of your labeled primer which may be much closer to reality than calculated one. And the difference between calculated and real annealing temperatures of labeled oligos may reach 7C or even more!
There exist at least one software that allows to predict the effects of fluorofores to hybridization parameters of oligos: Visual OMP. I tried it, but the predictions sometimes were not preсise
I use IDT for design of Taqman probes as well, and usually it works. But there occur some exceptions. See, for exemple, the paper attached:)
But I agree, calculated Tm (-5 to +2) may be enough for most cases
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line