Help with primer design through PRIMER3 - (Apr/03/2012 )
How I can design a intron spanning primer (forward primer on 1st exon reverse primer on another exon) or exon junction primer (primer spanning exon exon junction) through Primer 3.
Thanks in advance for help.
I don't have experience using Primer 3, but normally I use the web server of Invitrogen, they worked pretty well to me, and it's also user-friendly. Just put the sequence in the box, choose the region to be amplified, and change the condition a bit, then you will get several options.
Thanks for our suggestion, its working well.
I have ordered the primers and lets see how well its working
You can use PrimerBLAST, uses primer3 algorithm (just need to change default Salt correction formula and Table of thermodynamic parameters, to match those default in Primer3).
Or it's possible to design a exon-spanning primer by delimiting exon junction as a "target", primers would then be designed to have the junction between them. However you can do this only for a specific junction, not for any.
I used Primer blast, its giving well designed primers but for few of my target genes have very small exons flanked by large number of intron.
After cross-checking with UCSC PCR tool, its coming out to be not ok from exon junction or intron spanning primers.