I'm about to do RT-PCR soon, but I've been confused in regards to Primer design. Right now, the 4 genes I'm interested in are:
*Human IGF-1 (ncbi.nlm.nih.gov/gene/3479)
*Human TGF-Beta2 (ncbi.nlm.nih.gov/gene/7042)
*Human Beta-Catenin (ncbi.nlm.nih.gov/gene/56998)
*Human FGF7/KGF[ (ncbi.nlm.nih.gov/gene/2252)
I've had an initial Primer design (file attached), but I'm not sure whether these Primers are correct. My questions regarding Primer design:
* Many sites provide info in regards to gene sequence. Primers are basically short, complementary version of these gene sequence, designed to anneal during denaturation. Does it matter where it anneals? For instance, suppose a gene is 25000 bp long. Must the Primer I use anneal on the start of the gene (bp 1-50), or can it anneal on the middle of it (bp 10000-10050)?
* I tried using online Primer designing software (NCBI's Primer-Blast), but the program gave me more than one result. Are all these results okay to use, or should I be careful in choosing which one to use?
* In regards to NCBI' Primer Blast program, I tried using the reference sequence id (NC_000012.11 for IGF-1), but I always get "PCR template length (133851895) exceeded the limit: 50000. Please use primer range (i.e., forward primer "From" and reverse primer "To") to limit the template length. " error. Trying to paste the whole sequence resulted in the same error. I tried pasting just a portion of the sequence instead of all, and it worked; my question is, does it matter which portion of the sequence I used?
Thank you for helping me guys. I'm sorry if any of my questions are not clear enough; if there's such, I'd clarify my question for ease.