I have always done Q-PCR using ABI machines with TaqMan primer and probes designed by primer express.
I no longer have access to this software where I am. I designed primer and probes on IDT web site. There TM Calculation is very different than Primer 3.
I had to choose primers with TM of 55 and probe of 62 for a couple of my sets.
Is there any reason I should not do 3 temperature cycling program ?
Or should I stick with the two cycling program and lower the usual 60C down to 55-58 ?
Any opinions suggeations welcome,
Q-PCR using TaqMan Primer Probes
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