General guidelines for primer design

• 18-30 nucleotides

• G/C content: 40-60%

• Avoid complementary sequences of primers (especially at the 3’ end)

• Avoid mismatches at the 3’ end

• Avoid 3 or more G or C at the 3’ end

• Avoid a 3’ end T

Primer design to eliminate amplification from contaminating genomic DNA

Primer design to detect amplification from contaminating genomic DNA

Primer design and analysis tools

• Vector NTI

• Primer 3

• IDT SciTools

PrimerQuest

Oligo Analyzer

Real-time PCR

• QPCR

• Real-Time Vs Traditional PCR Post PCR processing

Poor agrose gel resolution (semi-quantitative)

Detect end point of the reaction

http://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf

http://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf

http://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf

ΔCt, ΔΔCt method to detect fold change