I think you would be familiar with Primer-BLAST. Its reliable and easy-to-use tool.

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

I always use Primer3. Easy, quick and we have always good and reliable amplifications:

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

@ Carlos -> Primer BLAST uses the primer 3 algorithm so its the same but inmy opinion Primer BLAST is easier to use..

Yes..........i m familiar with the tools you all are suggesting but for addtional requirements like adding a re site n then checking that the primer is efficient, or start codon orf incorporation...................primer blast wd not allow these features to be tested ...........

for gene expression analysis I advise you aganist insertion of exogenous sequences in the primers.

If your aim is cloning you don't need really software because your parameters are too

much mandatory.

Hi, I think you are going to have to use your creativity and dig a little to get this right. I like Primer3 (to get a list of possible primer pairs) and Perl Primer (to check for extendible primer dimers) and IDT Oligo Analyzer (to check for hairpins, etc...) (all free) I have also used primer blast...just check the primers once you do using the above sources. When doing gene expression analysis it is important that your amplicons span an intronic sequence. Using NCBI BLAST you can find these boundaries (its been too long for me to be able to describe this process, but it is possible). This is important because contaminating DNA can interfere with results, and since the mRNA doesn't contain introns, spanning those introns will remove/reduce the contribution from DNA...since amplifying long products is not as efficient as shorter products.

Use any primer designing tool example- primer3, pride, autoprime etc... select the best outputs and do blast. see to that your blast result should show only the target gene sequences... hope this information will be useful for you..

Cheers......

Jesu

Thank u one and all for ur inputs ...........!

Dear Niti

I always make use of the OLIGO Primer analysis software. With this you will be able to ajust the length of your primers, edit (add any wanted codon) the primers and check their efficient in the PCR reaction; you also directly have the optimum annealing temperature for your pcr process.

Hi Niti,

I think Invitrogen's Oligoperfect tool is better in your case

Thank u for ur input folks !!!

Primer 3 is more preferable tool. you can also use the software too of the seller from where you are planning to get your primer.

primer 3 plus with special settings for qPCR