Primerquest by IDT is excellent for this purpose.

http://www.idtdna.com/Primerquest/Home/Index

there is a video on the website which would guide you easily.

Just paste your sequence in Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) and click 'Pick Primers'. And order the primers through any company. That's it.

There are many options for primer designing (See the list of tools at: http://www.startbioinfo.com/oligos/ ). Manual primer designing is the best in my opinion (& I can take you through simple steps, if you want - via email). If you are looking for primers that are very specific to an alternatively spliced variant, primers from exon-exon junctions will help (a comparison of different tools for such specialized primers is available at: http://www.startbioinfo.com/junction-primers-comparison/). The comparison includes one of the software we have developed especially for mammalian species.

I think FastPCR softwares is good in this regard and it's easy to use. You can use the online version or download the latest desktop version. See attached brief software's review.

Article FastPCR © : Useful free software for molecular biologists Program

There are some good databases to use that already have the primers designed, all you have to do it order 2 or 3 sets from the database to test them out... http://pga.mgh.harvard.edu/primerbank/

http://primerdepot.nci.nih.gov/

Primer 3 http://bioinfo.ut.ee/primer3-0.4.0/

a) Reduce the size of product to the range 150 bp to 250 bp

b) Increase the GC content to 55 %.

c) Reduce max self complementary parameter to 5

Thanks all but what precaution have to use as some have dimer and hairpin loop and gibbs enery is -4 or -5

Another method, which is relative slow, but probably easy for a beginner:

step 1. to identify which ORGANISM (e.g. human) and which transcript variant if any is wanted, so you need go to NCBI nucleotide website and find the RefSef

step 2. go to "Pick Primers", which usually is on the right of screen and second under "Analyze this sequence"

step 3. set PCR product size and exon/intron selection, and click "get primers". Every selection has a help.

step 4. pick a pair of primers, which have smaller "self"s

Good luck

We generally derived primer sequences from qPrimer depot and validate by blasting in primerblast. I have obtained several primer pairs which can work in multi species.

Here are some online tool for PCR/ RT-PCR or sequence

1. UCSC Genome informatics (URL:http://genome.ucsc.edu/)go to PCR

2. PrimerBank PCR Primers for Gene Expression Detection and Quantification

(http://pga.mgh.harvard.edu/primerbank/)

3. RT primer by Roche and Biored (UR:http://www.rtprimerdb.org/)