Qiagen Assay Design Software is recommended for pyrosequencing. The software is available for FREE to our customers, to obtain a copy please contact us. We provide assistance with training and design of pyrosequencing assays. An assay design MUST be submitted to the PAN Facility prior to run.
- PCR primers should be 18-24 bases long, with anneal temp of 50-68°C.
- One PCR primer MUST BE biotinylated at the 5' end, and should be purified.
- Amplicon lengths should be between 50-300bp.
- Pyro-Sequencing primer must be in the opposite direction of the biotinylated primer
- Pyro-Sequencing primer should be 15-20 bases long, with annealing temp of 45-55°C.
- Pyro-Sequencing run should be less than 100 bases.
- Considerations for CpG Assays
- PCR primers should be 22-30 bases long, with annealing temp of 56°C.
- Pyrosequencing primer should be 18-23 bases long.
- Amplicons should be less than 200bp.
- For PCR, sample concentration should be ~50ng/rxn
- For bisulfite treatment, sample concentration should be 500ng-2ug
- DNA should be purified prior to PCR, but does not need to be purified before Pyro-Sequencing run.
- Please submit PCR and Pyro-Sequencing primers at a 5uM (5pmol/ul) concentration. Please provide ~5ul of primer for each each rxn.
- Online ordering is now available. Please click on "Place Order" in the menu bar.