2 replies to this topic

[daixiaol]

Hi, i am a beginner in rtpct.
I wonder how to know the primers span an intron within amplified region?  

[anonymous]

Hello,

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 ( [url="http://genome.cse.ucsc.edu/goldenPath/octTracks.html"][url]http://genome.cse.ucsc.edu/goldenPath/octTracks.html[/url][/url] ) 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.

Good luck.

[daixiaol]

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.