Sanger Sequencing

Guidelines

The LGTC facilitates Sanger Sequencing (template and primer pre-mixed) and has a price deal with the external company Macrogen. All sequences are processed on Applied Biosystems 96-capillary (ABI3730xl) systems.

First time users

First time users can ask for a short introduction course by an LGTC technician. Please contact info@lgtc.nl to make an appointment.

Sample requirements

The samples must be delivered premixed:
5 ul template + 5 ul primer (FINAL primer concentration = 1 pmol/ul) in a total volume of 10 ul.
Template concentration is size related. Recommended template concentrations:

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

Sample submission

Submitted samples must be scanned and delivered in the required tubes or else your results will not be traceable and you will not receive your data.

Rules:

Submission is possible throughout the day

All samples must be submitted in mandatory 2D barcoded tubes and caps. These are available at the LGTC outside office R5-20 and can be order via lgtc.nl/order

Submit the samples in R4-21 via the LGTC LIMS system, via lgtc.nl/order

Store your tubes in a rack in the TOP DRAWER of the -20 freezer in R4-21

REMARK:

Submitted samples must be scanned and delivered in the required tubes or else your results will not be traceable and you will not receive your data

Results

Samples will be collected for sequencing every day around 3 pm. Results will be delivered within 24 hours after pick up, unless indicated otherwise on our website lgtc.nl/order. 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.

The three stages of the sequencing submission are:

Submitted: Your samples are submitted and still in the freezer or have been picked up and are still being processed at Macrogen.

Checked: Your sequences have been split over different sequencing days/plates and not all is ready yet

Ready: You can download the zip file.

If after 3 days the status is still “Submitted” or “Checked”, please contact us.

Data analysis

You will always receive ab1 files. We recommend that you 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, this also means that the control taken along on all sequencing plates has met the ISO standard at Macrogen. This means that bad and/or failed reactions will not be repeated for free.
In all cases: It is your responsibility to make your own data backups. Please download your sequence data from the LIMS system within three months of submission, as we need to remove older data to prevent the system from crashing due to disk space limitations.
To view or to analyse 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 include FinchTV, Chromas, or Codon Code.

Cancellations

Once you have submitted your samples in the LGTC LIMS and they have been picked up, the request cannot be cancelled. We will charge you for the full price of the sequencing.