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Need help / questions about Primer design

Need help / questions about Primer design - Real-Time PCR and Quantitative PCR Forum

Need help / questions about Primer design - Real-Time PCR and Quantitative PCR Forum


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Old 01-06-2014, 08:07 PM
Pipette Filler
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Default Need help / questions about Primer design



Hello everyone.

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.
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File Type: txt Initial Primer.txt (390 Bytes, 0 views)
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Old 02-15-2014, 10:48 AM
Pipette Filler
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Default Re: Need help / questions about Primer design

Hi,
it is not that complicated as it may appear. First you can check literature if anyone already successfully used some primers for these genes and just find the sequences in material/methods. If you use oligo-dT for reverse transcription, the position of the amplified region should be close to 3'end. However, it is preferable to have at least one primer across an exon-exon boundary to prevent amplification of contaminating genomic DNA. I usually design the primers like this: The reverse primer is somewhere around stop codon. The forward primer is on the exon-exon boundary between the two exons preceding the last exon. The primers are 20-25 bp long and have 40-60% GC content and the amplicon is 80-150 bp long. If the sequences near 3'end do not allow such primer/amplicon properties due to e.g. extremely AT-rich stretches or too long exons, I move the amplicon a bit in 5' direction until it matches the criteria.
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