PAN_PYROSEQ

PAN: Protein and Nucleic Acid Facility

» Pyrosequencing

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Genomic Services

• Next Gen Sequencing

• DNA Sequencing
& Fragment Analysis

• Single Cell Genomics

Protein Services

Located in rooms
B065 and B017

• Sample Submission

** Please check for the appropriate sample submission requirements for each service type before placing your orders

Full Service- PCR and Pyrosequencing Run
Pyrosequencing Run Only
Self Service

Full Service - PCR and Pyrosequencing Run: (back to top)

• Assays/Primers Design (optional): using PyroMark Assay Design software.

• You can design and synthesize your own primers set and bring them to us along with your template DNA. You will need to email us your target sequence along with the assay file (.XML file).

• We can assist you in designing the PCR primers and the Pyrosequencing primer based on your target sequence. You will need to email us your target sequence and let us know the region of interest. We will email you the primers' sequences so you can have them synthesized and tested out.
** Please note: to streamline the process, we can also synthesize your primers at PAN. Once we got the primers, we will let you know so you can bring down your template DNA for the PCR step.

Criteria for primers set:

• PCR primers:

• One primer must be biotinylated and in the opposite direction of the Pyrosequencing Primer

• All primers should be purifired

* The optimal PCR amplicon lengh is between 80 and 200bp, although 500bp might work well for Pyrosequencing assays on genomic DNA.
* Region of Interest should be less than 100 bases.
* Avoid long stretches of homopolyers, especially next to variable regions (SNP/CpG sites).
* Avoid strong hairpins and primer dimers.
* SNP PCR primers should be 18-25bp, with annealing temp of 60-70°C.
* CpG PCR primers should be 22-30bp.

• Pyrosequencing primer:

• Pyrosequencing primer should be 15-20 bases long, with annealing temp of 45-55°C.

• Pyrosequencing primer should be located 1-5 bases away from first SNP or CpG site.

• Primer should be purified.

• Submission requirements:

• Template DNA: For each PCR reaction, template DNA should be submitted as follows:

* Concentration: ~10-25ng/ul
* Volume: 10ul

• Primers Set (for both PCR and Pyrosequencing): For each reaction, each primer should be submitted as follows:

* Concentration: 5uM/ul
* Volume: 5ul

Pyrosequencing Run Only: (back to top)

• Submission Requirements --

• Assay file (XML file) - Email us your assay (.XML) and/or run (.pyrorun) files.

• PCR product (biotin-labelled):

**Please note: PCR product should not be purified before Pyrosequencing run

* Volume: 20ul per reaction

• Pyrosequencing primer: for each reaction,

* Concentration: 5uM/ul
* Volume: 5ul

** After placing your order online, please bring your sample(s), primer(s) and a copy of the order summary to PAN, Beckman Center, Rm. B017.

Self Service: (back to top)

• Client prepares and runs samples on the PyroMark Q24. Consumables are not included but can be purchased from PAN. Please inquire.