PCR primer Design
In general, a length of 18–30 nucleotides for primers is good.
Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other.
If the Tm of your primer is very low, try to find a sequence with more GC content, or extend the length of the primer a little.
Aim for the GC content to be between 40 and 60%, with the 3' of a primer ending in C or G to promote binding.
Typically, 3 to 4 nucleotides are added 5’ of the restriction enzyme site in the primer to allow for efficient cutting.
Try to avoid regions of secondary structure, and have a balanced distribution of GC-rich and AT-rich domains.
Try to avoid runs of 4 or more of one base, or dinucleotide repeats (for example, ACCCC or ATATATAT).
Avoid intra-primer homology (more than 3 bases that complement within the primer) or inter-primer homology (forward and reverse primers having complementary sequences). These circumstances can lead to self-dimers or primer-dimers instead of annealing to the desired DNA sequences.
Primer Length
The optimal length of primers is generally accepted as 18–24 bp in length. Longer primers will take longer to hybridize, longer to extend, and longer to remove thus produces less amplicon. Primer Melting Temperature (Tm)
This is the temperature at which 50% of the primer and its complement are hybridized. To optimize for qPCR find primers of minimal length which have melting temperatures (Tm) that are between 59 and 68 °C, with an optimal Tm of 63–64 °C. Also, the Tm of the primer pair should be within 1 °C of each other. The primers should also have a Tm which is higher than the Tm of any template secondary structures (found using mFOLD software, discussed later). Annealing Temperature
Optimal real-time PCR annealing temperatures are 59 °C or 60 °C. Product Size
An ideal amplicon should be between 80 and 150 bp. If multiple genes are used, (i.e. comparing the relative expression of several genes) then the size of all amplicons should be close in length. SYBR® Green detection will produce a more intense fluorescence in larger products than smaller (so keep multiple products close in length). Mg++ Concentration
The default is set to zero on most primer design software. SYBR® Green buffer mixes contain 3 to 6 mM of MgCl2. Repeats
A repeat is a nucleotide sequence (a dinucleotide) that is repeated (e.g. TCTCTCTCTC). These should be avoided because they promote mispriming. If unavoidable, the maximum number should be 4 di-nucleotides. Runs
Runs are repeated nucleotides (e.g. TAAAAAGC has a 5 bp run of Adenine). Runs should also be avoided because they are prone to mispriming. The maximum run should be no more than 3–4 bp. 3′ Stability
This refers to the maximum ΔG of the 5 bases from the 3′ end of the primers. (ΔG is the Gibbs Free Energy, the energy required to break the bonds present at the 3′ end) A higher 3′ stability will improve the efficiency of the primer. GC Clamp
This refers to the maximum ΔG of the 5 bases from the 5′ end of the primers. Often called a GC clamp, the 5′ stability refers to how stable the 5′ end is due to the amount of Gs or Cs present at the 5′ end of the primer. Having 1 to 2 GC clamps are ideal, as it allows the primer to bind strongly to the template strand, making it more specific, however; avoid more than 2 GC clamps.
http://frodo.wi.mit.edu/primer3/
https://www.idtdna.com/scitools/Applications/RealTimePCR
http://onlinelibrary.wiley.com/doi/10.1002/bmb.20461/full
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
http://www.genomecompiler.com/tips-for-efficient-primer-design/
http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/
http://molbiol-tools.ca/PCR.htm