T3.4 What is wrong with these primer pairs?

Primer Sequence 5’→3’ Length %GC Tm (ºC)
Set1_Fw ATTTGGGGCCCCAAATGCTCA 21 52.4 73.3
Set1_Rv AGCTCGACCGTCGAGCTGAC 20 65.0 69.7
Set2_Fw ATTTAATCATTAATTTATTATTAT 24 4.2 47.9
Set2_Rv AGCTCGACCGTCGAG 15 66.7 59.2
Set3_Fw AGCGCCCGGACGGCAGGTC 19 78.9 78.2
Set3_Rv GCCGTCCAGGCGATGCAAC 19 68.4 73.8
Set4_Fw AGCGATCGACGAGTTTTTTT 20 40.0 63.0
Set4_Rv ATGCGACGCAGATACGACGCAGCTTTGCGATA 32 53.1 82.3


The primer contains an homopolymer
The primer is too long
The primer is too short
The %GC content is too low
The %GC content is too high
The Tm is too low
The Tm is too high
The primer can fold back and form a hairpin
Remember! Designing a good primer set is a complex process, but these minimal rules must be preserved;
  1. Length 18-22 nt
  2. Tm 52-60ºC (pair difference <2ºC)
  3. GC content 30-70% (pair difference not huge)
  4. Avoid hairpins
  5. Avoid self and cross dimers
  6. Avoid repeats (TATATATA, etc)