It is important that real time PCR primers are designed properly. Poorly designed primers will contain complementary sequences often binding on themselves and resulting in primer dimer and poor annealing to DNA template. Hence proper design of primers will save time, effort and money.
Follow the simple steps to design a primer
1. Go to Pubmed.com and enter the details of the gene of interest and choose "Nucleotide" in the drop down menu.
2. Look for the complete CDS or the mRNA sequence.
3. Copy the open reading frame sequence into word and highlight the regions between the introns and exons. Remember that when introns are removed the exons can be brought together. This is the region that is made into protein.
Alternatively, intron exon boundaries can be identified using a programme that compares the given genomic DNA sequence with the cDNA. Usually such a programme colours the 2 sections and then the introns can be removed.
4. Finally the PCR primes can be designed against 2 exon boundaries.