- The assay design software will design PCR primers and a sequencing primer for the region of interest.
- The SAEF recommends using this software as it generates primer sets (each primer set consists of a PCR primer pair and a sequencing primer) that fulfil the specific requirements for pyrosequencing analysis, and the nucleotide dispensation order.
- The SAEE is happy to assist with installation of software and training.
- DNA should be purified and of high molecular weight (i.e. good quality).
- The recommended amount of gDNA for a standard 25µL or 50µL reaction is a minimum of 10ng.
- For information regarding bisulfite modification kits, please contact the facility.
- One PCR primer must be biotinylated and HPLC purified.
- The biotinylated PCR product should be between 100 - 300bp.
- The pyrosequencing product (i.e. the region of interest within the PCR product) must be <100bp.
- Check the PCR product on 1.5% agarose gel. If there is a clear, strong product band (i.e. no excess primer, primer-dimers or other non-specific products), 5-10µL of the PCR should be sufficient for pyrosequencing.
- The SAEF recommends running 50µL PCR reactions (particularly if samples are to be run in triplicate).
- The SAEF requires the biotinylated PCR product, sequencing primer and the dispensation order (determined by assay design software).
- The SAEF will run the pyrosequencing reaction.
For more information regarding the sample preparation procedure, please contact the facility.