The secret to successful primer and probe design
Precise primer and probe design is essential for successful PCR reactions.
The optimum design parameters are the key to specific amplification and are set as default in our online design tools:
- PCR primer design
Specific design of primers for standard PCR, RT-PCR (reverse transcriptase) and DNA sequencing.
- qPCR primer & probe design
Optimal primers and probes for single plex real-time qPCR applications.
- Bi-Plex qPCR Assay design
Perform the design of primer and probe sets for 2 target regions for multiplex qPCR analyses.
- LightCycler probe design
Design of specific primers and probes for FRET assays.
The results are scored according to the best predicted performance criteria.
Selected primers and probes can be transferred directily to your online shopping cart
What you can expect from our primer design tools
Considered design criterias for improved specifity:
- Physical properties of the PCR primers
Optimum primer lengths, G/C as 3’-clamp, GC-content, primer Tm and Tm difference between both primers, repeats and optimal primer-primer annealing scores
- Physical properties of the PCR product
Best product length and GC-content range, primer Tm, repeats and optimal primer-template annealing scores
- Appropriate annealing temperature (Ta)
Avoiding primers having secondary structures and self dimer formation, building primer dimer formation and any non-specific binding sites on the template
- Thermodynamic calculations
- Primer melting temperatures are calculated by using the Borer nearest-neighbour model; thermodynamic parameters for DNA nearest-neighbour interactions; salt dependence of oligonucleotides
- PCR product melting temperatures are determined by using the formula of Rychlik, et al. based on formular of Baldino, et al.
- The output of the calculated PCR primer pairs includes a proposed annealing temperature (Ta) for each listed primer pair. The Ta is calculated using the formula of Rychlik, et al.
Hillier and Green; PCR Methods and Applications; 124-128 (1991); Baldino, et al.; Methods Enzymol. 168; 761-777 (1989); Rychlik, et al.; Nucleic Acids Res. 18; 6409-6412 (1990)