RT-PCR, help needed - (Mar/25/2002 )
Hi, i am a beginner in rtpct.
I wonder how to know the primers span an intron within amplified region?
You need to check your sequence to find out its genome structure. Some mRNA sequences in GenBank may have exon and intron information; some may not. In the latter case, you may BLAST your mRNA sequence againt GenBank genome DNA to find its gene structure. There is a convenient place at UCSC ( http://genome.cse.ucsc.edu/goldenPath/octTracks.html ) for you to check your gene structure. You can find your gene by typing your gene name or symbol into the search box.
Anyway, RT-PCR doesn't necessarily require primers to span introns.
i am using two-step RT-PCR this week and get very strong and good signal. The primers I am using have an intron with amplified region. I could know the pcr product is from the genomic DNA or mRNA transcripted cDNA.
I thought that two-step is much more better than the one step.
Thanks a lot for "pcrman"'s help.