9.3 years ago by
Sorry, I am really not an expert on this, but if nobody else can help, I just tell what I would do int this situation...
I think this is a good and very concise first read:
Actually, it is worth noticing that, most important parameters are not free-to-choose parameters but constraints. These constraints are defined by your real-time RT-PCR protocol. Say Tm, primer length, primer GC.
Read your protocol or machine manual or ask the person running that machine, or call the vendor, that will tell you most constraints, so the optimum constraints are those which are optimal for your protocol. Then use these optimum constraints as parameters. Say, the optimal Tm is 58 degr, set set Tm range to 57.5-58.5, size must be 20, set the size range to 20-20, if the optimal product length is 250, then.... Use the region which you want to amplify, maybe spanning an intron to be able to distinguish DNA contamination.
Then use primer3 web to iteratively try out these parameters. Leave all the parameters you don't know about as default. If you don't find any primer, relax the constraints a bit. The problem might be in general that with the optimal parameters you don't get a primer!
This is very important: After getting a primer pair, run a sequence search eg. using BLAT against the genome if available, and vector and contaminant databases. If the primers are not unique, try again.
I hope this helps.