Hello,

I would suggest a TM of 60°C for the primers and about 10°C higher for the probe. For designing primers and probes I would recommend this page:

http://bioinfo.ut.ee/primer3-0.4.0/primer3/

This page is really useful. Usually I only change the TM values and leave the rest as it is. Then I use BLAST to check the specificity of the primers.

Best wishes

Dirk

Dirk, thanks for your response. In a one-tube nested realtime PCR, there are two sets of primers (outer and inner) and one probe. I am asking which set primer's TM should be higher than the other set or it does not matter.

Sorry Zhenyu, I missed the word "nested". I guess that the probe should bind within your two inside primers. Maybe you can start with a lower TM for the first primer set for 20 cycles and then raise the TM for the second primer set and the probe for the next 25 cycles or so.

Thank you very much! This is also my thought.