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Design of Primer Probe Sets

Notes

  • Blast a sequence of the entire gene and identify a region which is specific for that gene.
  • After designing oligos do a BLAST search or a similar analysis to determine the specificity: www.ncbi.nlm.nih.gov/blast/blast.cgi
  • The length of the amplicon should be 80-120 bp in order to get high PCR efficiencies and more efficient 5’nuclease reactions.
  • The GC content should be 30-80% (ideally 40-60%).
  • Select the probe first and then design primers.
  • Place the probe as close as possible to the primers without overlapping them.

TaqMan Probe

  • Length
    - 18-30 bases
    - Optimal: 20 bases
    - Length over 30 bases is possible. In this case it is recommended to position the quencher not at the 3’end but internally between the18th and 25th bases counted from the 5’end (please inquire).
  • Melting temperature (Tm): 68-70°C. It must be 8-10°C higher than the Tm of the primers (8°C for genotyping, 10°C for expression profiling).
  • GC content: 30-80%
  • Avoid runs of an identical nucleotide, especially of 4 or more Gs (otherwise interaction between the nucleotides can cause the oligo to form secondary structure).
  • Select the strand that gives the probe more Cs than Gs (due to the strong interaction that G displays with itself).
  • Do not put Gs on the 5’end (quenches the fluorophore).
  • For multiplex assays (allelic discrimination)
    - Position the polymorphism in the centre of the probe.
    - Adjust the probe length so that both probes have the same Tm.
    - If very short probes are needed (less than 18 bases) please inquire.

Primers

  • Length:
    - 18-30 bases
  • Melting temperature (Tm): 58-60°C
  • GC content: 30-80%
  • Avoid runs of identical nucleotides, especially of 3 or more Gs or Cs at the 3’end.
  • The total number of Gs and Cs in the last five nucleotides at the 3’end of the primer should not exceed two.
  • Primers should scan exon-exon junction. Contaminating genomic DNA will not be amplified by these primers.

Primer Probe Design Service

  • Simply send us the accession number for your targets and let us design your primer probe sets for free*.
  • Please mention the desired 5’-fluorophore and 3’-quencher and contact us by email.
  • The delivery time for primer probe design and synthesis will be approximately 6 working days.
  • This offer is valid for up to 5 sets per week and customer.

 

*Free of charge service is limited to 5 designs per customer per day.

 
www.microsynth.ch
www.microsynth.ch