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Primer design

• Targets an amplicon length of 75 to 150 bp

• 50 to 60% GC content

• Limit secondary structure

• Limit stretch of G or C’s longer than 3 bases

• No stable interaction between forward and reverse primers (primer/dimer pairs)

• Place C’s and G’s on ends of primers, but no more than 2 in the last 5 bases on 3’ end

• Melting Temperature (Tm) above 50 oC

• Verify specificity

Probe design

• Scan gene of interest for g/c rich regions. They will have higher annealing temperatures than the average sequence and we want our probe to anneal about 8-10 degrees before our primers anneal.

• Find a likely probe sequence (has Tm of approximately 68-70 degrees) then go to the DNA folding site to see what kind of secondary structure this probe has.

• Please also be aware of what kind of probe you are designing.

• TaqMan probes should have a minimum of secondary structure to work well.

• Molecular Beacon probes have secondary structure in the stem sequences.You should calculate the probe annealing Tm without the stem sequence then add the sequence when you go to the folding site.

• After you have an appropriate probe sequence, design primers per the above rules that incorporate the probe