Genomics

Sanger Sequencing service

Complete Sanger sequencing procedure carried out by our facility

When the aim is sequencing a whole 96-sample plate with a single primer for all samples, the core facility  may perform, in an automatic way and without intervention from the user, the whole following protocol.

a)      When the starting material is PCR product: PCR product clean-up + sequencing reaction + sequencing reaction clean-up + run in the sequencer

b)      When the starting material is purified plasmid DNA: sequencing reaction + sequencing reaction clean-up + run in the sequencer

In this case, we have to be directly contacted (genomica@upf.edu). You may check the rates here. The user will provide us with the sequencing primer at a known concentration, and an estimation of the DNA concentration or PCR product amount.

When the sample number is below 96 or the user wants to perform the protocols himself/herself, the following steps should be carried out.

1.-  Sample preparation:

When the input material is PCR amplified DNA, we recommend to confirm a single PCR product presence by electrophoresis with an agarose gel. In this case, it is mandatory to perform the PCR product clean-up before carrying out the sequencing reaction detailed below. We offer the PCR product clean-up service for 96 sample plates (see rates).

When the input material is plasmid DNA, it is not necessary to perform this clean-up step and the sequencing reaction detailed below may be done directly.

1.1.-    Sequencing reaction (Sequencing reaction protocol).

Reagents: our core facility yieldsBig Dye Terminator v3.1 and related sequencing buffer (5X).

1.2.-   Sequence Clean-up (Sequencing reaction clean-up)

·         Sample in tubes:

When the number of samples is low (below 50), we recomend to prepare them in eppendorf tube of 1.5 ml.

Write down your user code (4 digits) and sample name on the eppendorf cap.

To speed up the sample processing, we appreciate sample name is short.

After sequencing reaction and sequence clean-up, deliver the dried sample (free of ethanol...) to the Service.

When the number of samples is 50 or more, we recommend to prepare them in plates.This system has some pros:

           • Automatized sample preparation: our Core facility offers PCR clean-up and sequence purification in plates.

          • Lower cost of sequences: the maximun volum of BigDye to be used is 1 ul for the sequence purification with Milipore (protocol)

          • Sequences are cleaner.

2.- Sample delivery to the Core Facility:

Sequencing applications will be filled in ONLINE (DNA sequencing application form). A copy of the ordrer will be generated, SIGNED by the user and handed to the core facility lab when samples are delivered for processing. Any application not generated by the online system will be refused.

At the entrance of the Service, right hand, you will find a little freezer for sequencing sample stock and a tray to leave the sequencing application.

Once processed, a facility responsible will contact you by email and resulting files will be transferred to a central server accessible from any computer.

All result files transferred to the server will be automatically DELETED every Sunday. When you need to get data from previous weeks, please ask us by email.

There are two methods to download the generated data:

a) Users connected to the UPF net (Novel) may download the files following this path:

§  Mi PC �� Unitat P (Folders en "Cm-c1ed\Cmd") �� SCT �� Genòmica �� Sequenciacio

b) The rest of users may connect with the web browser to this direction: https://webfiles.upf.edu/NetStorage

Log on as user: .sctweb.ext.cm.ed.upf  and password (ask to the Genomics Core Facility)

At the left part of the "folders", open: NetStorage; Home@UPF ; Genomica i Sequenciacio

Download and save result files to your computer.