I am assuming you want to design simple SYBR-green based assays and not multiplex assays with probes. And that you are interested in expression analysis. There are a lot of software online available, the one I prefer and used in my PhD studies was PerlPrimer (https://perlprimer.sourceforge.net/). It is reasonably straightforward in its implementation. Another decent option is NCBI's Primer-BLAST tool. You will need to follow couple of rules for primer design, i.e. for mRNA expression analysis I tended to prefer primers that span exon-exon junctions (which you can filter for in PerlPrimer). If you are doing SYBR-green based assay, make sure the annealing temperatures are similar between your reference and target gene(s). Prepare to have 2-3 primers out of 10 to not work (dimers, strange curves etc) with this route, but it is pretty cost-effective given how cheap primers are nowadays. For more complex assays (TaqMan, Scorpions etc), PREMIER Biosoft offers a commercial product if you have the budget (https://www.premierbiosoft.com/products/products.html).
Dear Saira Bano
Primer for which application?
- RT-PCR?
- Gene cloning?
- Serotype identification?
If you are interested in manual primer desing for gene cloning, you can found some tips in the following videos, present on my blog, ProteCool
https://www.blogger.com/video.g?token=AD6v5dzxz85z69as_qBJCTiisMtVv4q-Q0ChLHJHqSwNRA_FWtuB9p36qi4nQxd4b3OB2cxClbV6BmNDgkIdwfDUZqcDdOrGBSsjcuGTQKHAIEJPIrzIbIxwHrRRfZQf_UG0xZwwe2I
https://www.blogger.com/video.g?token=AD6v5dx7hIjOrRfLt70Lph2VJXXgyfu7CVeVUqMIzPNb5ySq3fHJF5QSXLTq93C-8iRHI_fT9_JfcwYQiGCHj0uYBMQzkB7DU8_qi34YSr9ZJzA7OYfHQav5-9eO6Idz5f06bydovl8
i hope this will be helpful to you
best
Manuele
Hi, Simple way to design Primers for Gel-based PCR process use Primer3 software from online or IDT Primer Quest Software
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