Primer Design

can someone tell me about the design of PCR primers in real time?

how?
Do I have to be careful, etc.?

There are computer programs and even web applications you can use to make this process much less tedious. Unfortunately I have a total mental block about the one I used to use. It was free and easy, so it's a shame.

You get the gene sequence that you want to amplify (or cDNA sequence rtPCR if you do), then plug it into the program and put in your settings.

You are looking for normal primers that amplify a segment of ~ 200 base pairs, give or take a little.

They must be unique and not free for any other gene in the species you want to (check this with BLAST search after the program hands you some suggestions of primers.)

The primers should have melting temperatures within a few degrees of each other for better performance. If you have a set of primers for GAPDH enzyme or other maintenance that you will use as a control, make sure they will all work with the same annealing temp.

Primers should not have regions of complementarity within a single primer that is more than three base pairs that could anneal together and form a hairpin loop in your primer. Clearly, they should not be complementary to each other, either.

It is good if the end of the primers G or C.

I think we used to be between 18 and 25 bp.

Since oligos are cheap, we used to always three pairs and test everything on a model we know is good with regular PCR and make sure we get a good group and choose the most suitable one to avoid wasting money, real-time PCR with a primer for some reason does not work even if it seems like it should.