Despite finding many websites for primer design in promoter regions, I am in need of guidance from someone experienced to direct me through the appropriate steps.
This is going to depend on your organism, gene(s) of interest, and how conserved promotors are in your organism's genome. Talk to folks in your lab. The general guidelines for one organism (e.g. Arabidopsis) do not necessarily apply to other organisms (e.g. maize).
Katie A S Burnette Thank you for your informative response. Your advice is invaluable.
In general start trying to clone 1-3kb upstream of the start codon and a sequence that includes the first exon for the gene (some species have enhancers there). Try and design your primers so that the 5' ends are at GC rich motifs with low polymorphism. Aim for primers that are 15-30 bp long and ideally have a similar melting temperature (not always possible so use the lower one for the PCRs). ALways order in several primer pairs because some will not work. Some species have alot of secondary structure in their genomes so you may need to use a high GC content PCR protocol even if your target region is low in GC content.
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