Sequencing via capillary electrophoresis
Sanger Sequencing via the LGTC
The LGTC facilitates Sanger Sequencing (template and primer pre-mixed) and has a price deal with an external company (Macrogen). All sequences are processed on Applied Biosystems 96-capillary (ABI3730xl) systems. The ABI3730xl is equipped with 50cm capillary arrays and use POP7 polymer as a separation matrix.
We offer the long run Sanger sequencing service which reads approx. 900 base pairs. We combine all samples in 96-tubes racks in the TOP DRAWER of the freezer in R4-21. All Sanger Sequencing samples must be submitted in dedicated 2D barcoded tubes and caps. These tubes and caps are provided by the LGTC. No 96-well plate will be accepted. Without 2D barcode we will not be able to trace and dispatch the data.
Sequence samples can be delivered at any time. The 2D barcoded tubes and caps are available at the LGTC in R4-21. At the same location you can scan your barcoded tubes and submit your samples in the LGTC LIMS system. Once your samples are submitted in the LGTC LIMS system you can add them in a rack in the -20 freezer, also located in R4-21. Always put your samples in the racks in the TOP DRAWER. A full manual on how to submit and deliver your samples can be found here. If you add samples in the rack without scanning the barcodes, the data will not be traceable and thus cannot be delivered. We will charge you for the full price for the sequencing.
The samples must be delivered in 2D barcoded tubes and caps (mandatory) with the following template and primer mix, already pre-mixed. A total volume of 10ul is enough: 5ul template_concentration + 5ul primer (FINAL primer concentration 1 pmol/ul)..
Recommended Concentration and Volumes for different size templates:
- BAC concentration over 500ng/ul
- Plasmid (over 4kb) concentration 200ng/ul
- Plasmid (below 4kb) concentration 100-150ng/ul
- Purified PCR product (over 700bp): higher than 50ng/ul
- Purified PCR product (300bp ~ 700bp): 25-50ng/ul
- Purified PCR product less than 300bp): 10-15ng/ul
First time users
When you have samples for Sanger sequencing and you do not know how to proceed, you can make an appointment at the LGTC. We can show you how to deliver your samples and all other steps. For large projects or series of samples it may be wise to start with a few pilot samples to optimize the concentration of template and primer for the sequencing reaction.
Once you have submitted your samples in the LGTC LIMS and they have been picked up, the request cannot be cancelled.
Availability of sequence data
Samples will be picked up at the LGTC freezer every day around 3 pm. Results will be delivered within 24 hours after pick up. When your results are available you will be notified by email. You can also check the availability of your sequences yourself by logging into the LGTC LIMS System. Always check the LGTC LIMS system before contacting the LGTC to ask if your sequences are available. When technical problems occur that might jeopardize the delivery of your sequence results within 2 working days, the estimated delay of the sequence service will be mentioned on our home page. If such a delay is problematic for you, please contact the LGTC to discuss alternatives. When after 3 days the status is still “Submitted” or “Checked”, please contact us.
The four displayed stages of the sequencing submission are:
- Submitted: Your samples are submitted and still in the freezer or have been picked up and processing.
- Checked: Your sequences have been split over different sequencing days/plates and not all is ready yet.
- Ready: You can download the zip file.
Data and data analysis
You will always receive ab1 files. We recommend you to check the raw data since sequences with raw data intensities below 500 are not guaranteed by Applied Biosystems or by the LGTC, and we cannot exclude capillary cross talk. Please keep in mind that when we receive data, the control taken along on all sequencing plates, meets the ISO standard. Thus bad and/or failed reactions will not be repeated for free. In all the cases: we cannot guarantee that we keep your data for longer than six months, it is your responsibility to make your own back ups. Download your sequence data from the LIMS system within three months after completion; we need to remove older data to prevent the system from crashing due to disk space limitations.
To view or to analyze your ab1 files we recommend Sequence Scanner (free to download from the Applied Biosystems website). It is a good software to view traces and raw data.
Other options are FinchTV, Chromas, or Codon Code.