Primer Design

The best tool for designing primers is the Primer Express Software, which is sold by Applied Biosystems.  Nearly 90% of the primers I have designed using this software are acceptable.  A copy of this software is available in the UMDNJ sequencing facility for public use.  In my experience, other primer design programs, which are used for typical PCR, are not acceptable.  Alternatively, you can search the literature for published primers.  About 50% of the primers I use from the literature were successful.  Some primers are published on a public database [here].  

Now is a good time to talk about TaqMan versus SYBR green as technologies for measuring your cDNA.  SYBR green is cheaper, because it only uses regular primers.  Typically, these primers can be ordered for $10 per pair.  Another alternative is to design TaqMan primers.  These are designed using a free program on Applied Biosystems' website.  Although more expensive than the SYBR Green method, their primers are guaranteed to work (or they will give you another pair to try). Finally, if you can find TaqMan primers in the literature, you can order the central dual-labeled probe directly from your primer supplier, as a FAM and Black-hole quencher labeled probe, instead of ordering from Applied Biosystems.  Of course, Applied Biosystems doesn't guarantee that this method will work. 

To order TaqMan style primers, find your sequences in publications, or design them using the Primer Express software.  You will be ordering three separate DNA sequences:  two primers, and one internal probe.  The internal probe is a dual-labeled probe.  You can either order this through Applied Biosystems, or order a dual-labeled probe from a company, such as IDT for approximately $200.  Order a 5'-FAM and 3'-Black Hole Quencher to match a TaqMan style approach.  

So when is it good to order TaqMan primers?  If cost is a concern (like it is for most academic labs), order TaqMan style probes only for genes that you will measure often.  If you are just exploring to find novel targets, use SYBR green because it is cheaper.