Sample Preparation

 Assay Design

• 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. 

 gDNA Extraction

• 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.

 PCR

• 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).

 Pyrosequencing

• 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.