Hi Cheshta

I am using PRIMER3plus... Just put your target sequence that you want to amplify and the software will do the rest. You can adjust the size of the amplicon (around 150-250 bps is OK) and other parameters. The software will give you a first suggestion and several more depending on the target sequence and parameters.

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

Hope it helps

Cheers. Paco

Dear Chesta,

You can have a first look on NCBI web site for Aspergillus_fumigatus EST / cDNA (http://www.ncbi.nlm.nih.gov/gquery/?term=Aspergillus_fumigatus).

If you do not find your gene of interest you can look at the whole genome sequences (http://www.ncbi.nlm.nih.gov/genome/?term=Aspergillus_fumigatus).

Once you have retrieved the cDNA sequence of the gene of interest for A. fumigatus, you can design RT-PCR primers from available softwares (e.g. primer express, Applied Biosystems).

Hope it will help!

Fabien

Thanx alot (Francisco Enguita and Fabien Lamaze) for the prompt reply...

But Fabien: I am not able to find the complete cDNA sequence for A. fumigatus on the link u gave...I am getting sequence for Partial mRNA only (which I think does not have 5'UTR and 3' UTR)......can u please tell how to proceed?

Thanx....

Cheshta

Dear Cheshta,

Only partial mRNA of 200-300 bp could be enough to design Q-PCR primers, as the target amplicon size for Q-PCR will be around 60 - 100bp.

You can have a look at the link below if you want longer sequences:

http://www.ncbi.nlm.nih.gov/gquery/?term=Aspergillus_fumigatus

,then select protein sequence database.

You will have to retro-translate the protein sequence into mRNA sequence.

Hope it helps!

Fabien