I have been given some pre designed primers for performing qPCR and i have done the BLAST and have found that all the primers are specific to mRNA.
However i would like to check that whether my primer spans any exon intron or exon exon junction.
I have gone through many available suggested ways to do so but i am unable to figure out how to do it step wise.
Hence please guide me on this note.
Thanks in advance.
If you have the cDNA code in a FASTA format, you should be able to set BLAST to align your primers to the sequence. If they align, you've no introns.
Hi
the easiest things are often the best....put in the same file sequences from genome and mRNA, search your primers and you'll find.
fred
Finding primers specific to your PCR template (using Primer3 and BLAST).
This is how I do it: First with Primer-Blast find out what is the mRNA reference sequence, then open the reference sequence, and with the Pick Primer function (using Primer3 and BLAST) you can detect the location of the primer and if it is crossing an exon-exon junction.
under the UCSC "in silico PCR" tool (http://genome.ucsc.edu/cgi-bin/hgPcr), doing the PCR and going to the genome browser will ensure you to see exactly where your primers are spanning.
all the best
fred
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