One of the best site for primer designing would be 'Primer Blast' at NCBI site.....you can evaluate different primer pairs for a variety of interactions and also specificity for a particular gene....go ahead and try it..
I recomend you the program PerlPrimer because it is free, easy to use and it designs primers for standard PCR, bisulphite PCR, real-time PCR (QPCR) and sequencing.
Integrated DNA Technologies (IDT) has online tools on their web site (idtdna.com) that I've used for years. Their PrimerQuest tool is excellent and FREE! There is no obligation to order the primers from them but I can't see why anyone would order from anywhere else.
Another source is called PrimerBank (http://pga.mgh.harvard.edu/primerbank/). These are pre-designed for many genes and the supporting information is really nice for the loci I've looked at. I've tried one set of primers suggested by this site and they worked perfectly.
I allways had extrimely good results using Oligo 3 then Oligo 5. Now there is a 7.0 version and I never tested it but there is no reason that it underperforms as compare to the former ones. The tool is not free, but it deserve it.
Snapgene is a really easy and complete software to design primers and do other molecular cloning strategies. You can try the 30 days trial. http://www.snapgene.com/ And then you can check primer properties using Primer-3 or Oligo 3.
Netprimer picks up a lot of potential primer-dimer problems that primer3Plus misses, having a simple algorithm. However, I was somewhat surprised today, seeing a primer pair where the Tm for the left primer was 3°C higher than that for the right primer according to Primer3Plus and the MWG order system, but the Tm's were reversed (right one higher than the left one) when using NetPrimer. Are the calculations really that variable? We have used the Primer3 Tm output for >100 primer pairs with good results in qPCR.
IDT's PrimerQuest is the best tool there is. It is easy to use, has multiple options including the ability to change parameters, and, best of all, it's FREE. The search algorithm is very fast and is based upon years of research in DNA thermodynamics. The output is easy to interpret and you can select from among many possible primer pairs from the same output.
I agree with everyone that Primer3 gives a melting temperature difference of 3 degrees. I used Primer Blast in the NCBI website upon Daniel P Potaczek's recommendation and it provides one with more specificities. I would suggest Primer Blast instead of Primer3.
PrimerBLAST would be a good choise at first instance for generate a list of potential primers, then you can use OligoCalc http://www.basic.northwestern.edu/biotools/oligocalc.html
to check for secondary structures in the primers and mFOLD http://mfold.rna.albany.edu/?q=mfold
or UNAfold from IDT web page https://www.idtdna.com/UNAFold
to check for secondary structures in the product so you can decide which pair fits best for your experiment. There is an useful paper that guides you in the designing of primers for expression studies using free tools available on the web: Thornton B and Basu C. Real-time PCR (qPCR) primer design using free online software. 2011.Biochemistry and Molecular Biology Education 39(2):145.
To adequately access your question I would have to know what kind of instrumentation you have access to. I will try to give you my best advice if you contact me at [email protected]. That is a closed system, so no other peers can see what we are discussring. You are free to post any of my comments here afterwards.
Hi every one I appreciate each suggestion here. IDT's PrimerQuest is good tool and simple to use, furthermore many programs for primer designing and sites like MPE, UCSC
Depending on your application I would suggest you first use software for doing a multiple alignment, then use the consensus sequence as input for primer design with http://primerdigital.com/fastpcr.html
If you just have a very conserved sequence from an eucaryotic organism you can skip the first steps and just use the squence directly in the fastpcr-application.
You also have the option to use the free primer design tool at NCBI: https://www.ncbi.nlm.nih.gov/tools/primer-blast/
I've made a list of great free primer design tools previously (http://toptipbio.com/free-primer-design-programs/), these are:
Primer-BLAST (my personal favourite)
Primer3
Primer3Plus
PrimerQuest
OligoPerfect
PerlPrimer
OLIGO
GenScript Online PCR Primers Designs Tool
AutoPrime
RExPrimer
BatchPrimer3
Eurofins Genomics' Primer Design Tools
Personally, I prefer Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) by NCBI. This will simultaneously design primers and BLAST them against the genome for potential unspecific binding (this is key!).
I have also made a guide on how to design real-time PCR primers (for SYBR Green application) using Primer-BLAST (http://toptipbio.com/real-time-pcr-primer-blast/). This includes some considerations for optimal primer design. I hope it is clear enough.
I concur with Steven Bradburn on the use of Primer-BLAST in designing anf filtering out nonspecific binding as the NCBI GenBank database is rich in sequences deposited. But also Primer-BLAST incorporates Primer3 tool making it even more powerful.
The list of primer design tools introduced by Steven Bradburn is interesting and diverse. They could be useful for different purposes. Thanks for your full comment.