PCR Primer Design
Reagents
- PRIMER software.
I am using PRIMER software to design most of my primers. You can also use Primer 3 at http://frodo.wi.mit.edu/. There are several points you might want to keep in mind for primer design.
1. For cloning primer, there may not be a lots of choices but starting (ATG) and ending (TGA). Thank the advanced technology of polymerases, PCR amplification is usually not a problem for most of the targets even directly copying any sequence as primer.
2. When the PCR reaction does not work using copied sequence from start codon region and stop codon region, I usually design another set of primers 5\' up the start codon and 3\' down the stop codon for nested PCR.
3. For realtime PCR, the primers usually require more careful design. The product is usually 200 bps. The GC percentage for the primers themselves AND the product is usually ~50%.
4. With the PRIMER software, I first define the criteria for primers including GC%: 45-55%, length: <22 bps, complementary sequence on the 3\': <3 bps, complementary sequence anywhere within the primer: <7 bps.
5. I also try to keep the GC% of the PRODUCT at ~50%.
6. I usually try to avoid GC rich or AT rich regions not only in the primer but also in the product. I like balanced region as the target.
7. Your DNA sequence is usually bigger than 200 bps. One easy way to identify a good region is to walk through the sequence using PRIMER software. In any given region, if I can find a lot of qualified primers, I usually choose that region for target.
8. Finally, after primer design, I usually compare the primer sequences using BLAST in NCBI to make sure they can only amplify the product I want instead of close homologs or orthologs. To do this, simply paste the two primers into the input region of BLAST and choose NR (nonredundant) as the pool and perform a BLASTn. If all the 100% hits are from the gene you are interested in, these primers are good to go. If some 100% hits are not the gene you are interested in, find another pair of primers to avoid false positive results.
9. I order my primers fron Invitrogen. I dissolve the primer to a final concentration of 100 uM with H2O. I usually make a 10 fold dilution and keep the 10 uM primers for daily use.