I am working on gynoecy and parthenocarpic expression in cucumber where gene sequence information is available. Now looking for the ideal marker and approach for designing the primer.
Important things to keep in mind when designing primers include:
Designing pairs of primers with similar melting temperatures that are roughly between 60-70C.
An appropriate GC ratio (around 50%)
Avoiding dinucleotide repeats or mononucleotide runs (e.g., AAAAA)
Avoid primer sequences that would allow for binding within or between primers.
These and other general guidelines can be found here: (http://www.lifetechnologies.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.htmlhttp://www.lifetechnologies.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/primer-design-tools.html)
I also recommend the Primer3 tool for developing primers. It takes all of these concerns into account. (http://biotools.umassmed.edu/bioapps/primer3_www.cgi)
Best of luck!
Hi Thayyil Pradeepkumar
In addition to the valid points given by Weston.
For primer designing the primer length should be 18-20bp.
For designing the primers. Include some genome region outside the gene of interest to be amplified. So as to get the best possible set of primers to completely amplify the required gene.
You can use this link below. But my offer design alternative primer.
http://simgene.com/Primer3
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