Sybr green primers

The advantage of using Sybr green is that there is no special primer design necessary. You can use primers that you are already using. The best data will result if your primers are highly gene specific and you have optimized them for the annealing tempature that gives the best yield with the least amount of background.

Taqman primers and probes

Primer and probe design uses Primer Express software from ABI. We have both Mac and PC versions available on CD.

The input for primer/probe design is the DNA or cDNA sequence(s) you are trying to detect. If you are going to do real time RT PCR, the input is the cDNA sequence for the transcribed gene. In this case, it is ideal if you know the location of the exon/intron junctions, so you can locate the probe (or one of the primers) across this junction. This prevent amplification of genomic DNA decreasing any background signal. While it is possible to DNAse your RNA, this requires heating the RNA for sometime with an enzyme (something to be avoided with the ever fragile RNA).

There is a sheet from ABI (pdf) which takes you through primer/probe design and optimization. If you want to locate the probes on the exon/exon junctions of your RNA, you need only to mark the junctions in the initial page of Primer Express using the junction key (left side of screen). The software then knows to put the probe on one of the junctions you have marked.

Primers can be ordered from anyone. Probes are dual labeled oligos which must be HPLC purified after each label is added. These are therefore expensive. ABI will make them though other companies (Operon) are now offering them at a more reasonable price ($200-250). You don't get a huge quantify of probe, but enough for 1000 or more real time reactions.

Probes should be 5' labeled with 6'FAM and 3' labeled with TAMRA.