I have to design a probe for qPCR laying between a pair of primers that I know is working quite good (with sybr green). I have a couple of questions about it:
1) Would it be ok if there is no free space between the 3’-end of the probe and the 5’-end of the reverse primer?
2) The Tm difference between the primers pair and the probe is about 6°C (primers ~62°C; probe ~68°C). Is it enough?
All the other conditions for the probe that I am aware of are more or less fulfilled: no repetitions of more that 3 nucleotides; nor G nor C at the 5’-end; length: 28 nucleotides; low self complementarity.
What do you think about it?
Thank you all