I have to design a probe laying within two primers that I know are working quite good. I have some questions about it:
1) Is it ok if there is no free space between the 3' end of the probe and the 5' end of the reverse primer?
2) The difference of Tm between the primers pair and the probe that I have found is about 5-6°C (primers:~62°C; probe: 67.77°C). Is it enough?
All the other rules that I know are more or less fulfilled: no G or C at the 5' end; probe length: 28 nucleotides; no repetitions of more than 3 nucleotides; etc.
What do you think about it?
Thank you all