now a days it is always good to pick up published primers from reputed journals, if you dont find any, then,

check out the sequence of your gene of interest from NCBI database

submit that sequence in primer3 tool, go for pick primer option.

you will be provided with a set of primers

check those set of primers for primer quality like

1.primer dimer formation,hairpin loop formation(there are online tools to find out)

2.annealing tempereature( difference between forward and reverse primer should not exceed 5degree, and annealing temperature should be between 48-63 ideal is 52-58)

primer sequence should not be less than 17bp and not exceed 25bp(less than 17bp non specificity,more than 25bp high annealing temp)

3.GC content should not be less than 45 and more than 60.

once you pick the good primer cross check the primer using UCSC browser, or ncbi nlast tool.

You can try to use Prime 3 to design your primers, it worked for me (see the website: http://primer3.sourceforge.net/). I have also used the Primer3Plus (see website: http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/).

Good luck!

Best regards,

Ana M. Pinela

Hi, have a look here: http://oomyceteworld.net/protocols/primer%20designing2.pdf

I also use primer 3.

Cheers, Nadine

Yes there is software available, primer3 has already been mentioned. How good is your basic knowledge about this process, you need at least some of it to choose working primer pairs (please let us know so you can get some information).

One suggestion is go to Pubmed to find out the sequence of your interest then on the right side of the screen there is a place to click to choose the primers. It isalso based upon Primer3 but the system will run the blast automatically. This will provide you several options and you can select the one suit you most. For me, not a molecular biologist as trained it offer the most reliable source.

I design primers by first looking up the gene of interest on ensemble genome browser. Bring up the sequence and choose the exons of interest. Then paste that sequence into primer3 making sure to change the parameters to suit optimal amplification (you can google what a good primer pair should be) then once you have your desired sequence use net primer to make sure that no dimers or hairpins are forming and change the sequence as appropriate. Once you have a good pair you should then blast the sequence to make sure you're amplifying what you should be and not some random gene. If you've never designed primers before be prepared for some headache but it is the cheaper option compared to the pre-designed option. Also, if you're using these primers for qPCR don't forget to check primer efficiency by doing a serial dilution.

You can use UCSC Genome browser to design the primers.

http://genome.ucsc.edu/

Thank you for all, for your answers i got more information's.

Primer 3 is fine

i can make the primer through Primer 3 program.... try it well be effective

Primer3 is the best, but you also need to know what purpose of this primers is only ordinary pcr or for example real time pcr.

You can buy Geneious Software, use it for all bioinformatic work, starting from primer design to align etc...

please check this web page http://molbiol-tools.ca/PCR.htm

You can use Primer 3 to design your own primers http://primer3.ut.ee/ or "Vector NTI" application to do it.

For me, I often design "by hand": select a fragment about 20-30 base at the upstream and downstream of the sequence that I need to amplify. Then calculate the Tm, error,... of these primers by using online tool "PCR Primer Stats":

http://www.bioinformatics.org/sms2/pcr_primer_stats.html

You have to spend more time by above way, but you can select the primers at the expected positions.

Good luck.

now a days it is always good to pick up published primers from reputed journals, if you dont find any, then,

check out the sequence of your gene of interest from NCBI database

submit that sequence in primer3 tool, go for pick primer option.

you will be provided with a set of primers

check those set of primers for primer quality like

1.primer dimer formation,hairpin loop formation(there are online tools to find out)

2.annealing tempereature( difference between forward and reverse primer should not exceed 5degree, and annealing temperature should be between 48-63 ideal is 52-58)

primer sequence should not be less than 17bp and not exceed 25bp(less than 17bp non specificity,more than 25bp high annealing temp)

3.GC content should not be less than 45 and more than 60.

once you pick the good primer cross check the primer using UCSC browser, or ncbi nlast tool.

I agree with Artur Burzynski; also a simple search for "primer design" on this website will retrieve similar questions and very useful answers.

https://www.researchgate.net/search.SearchPosts.html?query=primer+design&accountHitCount=701&topicHitCount=3&postHitCount=17087&pubHitCount=3190&jobHitCount=447&institutionHitCount=195&departmentHitCount=3437&dbw=true

Hi Marimuthu, if you have a genome to work from, designing primers is pretty easy. I always start by downloading sequence of the desired amplicon from NCBI or from Ensembl.org. Next I put the sequence into Primer3 on the MIT Bioinformatics page. There are many settings there to play with but the only one that I usually adjust is amplicon length. The amplicon length will be different for various applications such as PCR, qPCR, cloning, etc... Use the information provided by the manufacturer or a lab mate for determining ideal amplicon length for your downstream application. Primer3 is great in that it takes care of self-complementarity and other issues. After coming up with a few candidate primer sets, I always use Ensembl to BLAST the primers back to the target genome to be sure they are specific to just the sequence I am trying to amplify. If you do not have a genome published to use. Look for sequence just for your region of interested published by others. If both of those are absent, you will have to design degenerate primers based on sequence homology to the most closely related species with a published genome. Hope that helps!

I think the best way to use primer 3 in NCBI. Its very effective as i seen

Primer BLAST in NCBI is the easiest and good one for designing primers. the link is given below.

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

Designing primer depends on which method you are going to use further ,whether Restriction -digestion based or restriction free cloning.For eg. In restriction free cloning the primer contains both vector and gene specific sequences with appropriate restriction sites while in Restriction digestion based cloning, the primer contains only gene specific sequences with restriction sites.I have used the Primer3 free online tool

http://simgene.com/Primer3

Good luck.

Try one of the following :

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

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

Use Beacon 5 soft ware, it is little expensive other wise you can use primer 3. This is free

the best one for primer design is NCBI database

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

It depends on choice, both primer3 and NCBI are good. But I think primer3 is more flexible.

Good luck

Thanks for all for this useful information.

Primer BLAST in NCBI is the easiest and good one for designing primers.

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

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

Although the post is very old, it is really useful for me. Thanks all for your comments