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.

http://toptipbio.com/free-primer-design-programs/

http://toptipbio.com/real-time-pcr-primer-blast/

https://www.ncbi.nlm.nih.gov/tools/primer-blast/

Http://frodo.wi.mit.edu/ this is a great place to start.

Hi Aadeshkumar

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..

bye

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.

I am using Primer3Plus, quite easy to handle.

http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/

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.

Primer3 series

http://www.basic.northwestern.edu/biotools/oligocalc.html

Try this also. works well.

Try this also. works well.

http://www.basic.northwestern.edu/biotools/oligocalc.html

Thanks to all

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.

Primer-3 is a very good software for primer design, tm estimation and blasting

As mentioned above, primer3Plus and PrimerBlast are both quite easy to use and give good results.

I prefer to primerblast.

Primer-3, PerlPrimer and PrimerBlast are most easy and user friendly

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.

visit the following website:

http://www.oligo.net/

Primer blast is best it works on same basics of primer 3. Dont use primer3Plus it gives lots of secondary structures

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. 

A high throughput web application for PCR and sequencing primer design

try to to use this one: 

http://probes.pw.usda.gov/batchprimer3/

I sow that many people use the Primer 3 software. I used it many times to design for SNP and InDels. However,  the Tm provided by this software is not always the best one.  

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.

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

http://bioinfo.ut.ee/primer3-0.4.0/primer3/

well sharing my file...

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/

You can use BiSearch server at http://bisearch.enzim.hu/?m=search

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.

http://toptipbio.com/free-primer-design-programs/

http://toptipbio.com/real-time-pcr-primer-blast/

https://www.ncbi.nlm.nih.gov/tools/primer-blast/

Find more answers at the following link on ResearchGate

https://www.researchgate.net/post/How_to_design_a_primer

here is a list of 13 Free PCR Primer Design Programs

https://toptipbio.com/free-primer-design-programs/

i hope this would help