Tips to design PCR primers

Guidelines for designing primers/probes for Real-Time qPCR

Goals of primer design

Maximize specificity (ie. Amplification of single product)

• Avoid mismatches/mispriming
• Avoid formation of primer-dimers
• Ensure primers are target specific

Maximize efficiency (ie. Complete synthesis of target each round of PCR)

• Avoid secondary structures within primers and amplicon
• Avoid formation of primer-dimers
• Avoid amplification of non-specific product

General design guidelines for primers

• Primer length should be between 18 - 30bp
• GC content should be 30-80% (ideally 50-60%)

..... (to be continued)