I hope that you can help me (yet again!)
I am new to RT-qPCR, and I'd like to ask some questions about the primers:
1) Where do you usually order your primers? Does it matter? What level of purification do you usually order for your primers - HPLC, PAGE or just standard desalting? I am following the protocol from T. Nolan (Nature Protocols 2006, 1(3), 1559), and it says that the primers have to be either HPLC or PAGE purified, whereas a sales representative at IDT DNA told me that standard desalting is fine (although it implies that a typical primer of about 22 bases or so will contain about 17% trancation products.
2) When you are starting a new assay, how do you usually get primers? Do you design them yourself, or do you look in the primer databases or literature to see what other people used? What online programs would you recommend for primer design?
3) When looking in the primer databases, I have found separate primer sets for SYBR Green and Taqman assays. Except for the presence of the probe, how would primer design be different for these two assays? What would be the difference in primer design if I am just interested in an RT-PCR assay (semi-quantitative?) - would the amplicon length be typically bigger?
Thank you very much for your help.