The GTL offers a comprehensive DNA sequencing service with a comprehensive range of services.
We are sequencing according to the Sanger method and use the "cycle sequencing" technology using labeled dye terminators (BigDyes). With the help of this technology, it is possible to sequence purified PCR products, plasmid DNA and BACs. We offer different read lengths and analysis types (see below). Furthermore, we have a variety of special protocols in order to be able to sequence even difficult templates.
Samples that are submitted to our lab before 10:00 am are usually processed the same day and results are made available for download the following day. We can also analyze particularly important and urgent samples within 4 hours (see Xpress service).
Several ABI 3130XL Genetic Analyzers from Applied Biosystems are available for analysis. Sequence data analysis is carried out with the Sequencing Analysis Software (Applied Biosystems), so the quality of the data can be assessed.
If you are planning larger DNA sequencing projects, please contact us. We are always ready to provide further information, assistance with problems related to Sanger sequencing.
Only 'Long-Read' or 'Short-Read' analyzes can be performed per job! Please do not mix!
Important: All your DNA samples must not exceed the security level S1.
For further questions you can us.
The following options are available for Sanger Sequencing
FAQ - Sanger Sequencing
In order to be able to use our DNA analysis service, you have to register in our TSM system.
We need the following documents:
a) Before submitting your first order, we need your consent to the data protection declaration. This happens automatically during registration
b) Written order placement, e.g. in the form of a "Blocvkzettel" or an internal order form. Please enter the number of samples requested, the desired read length (approx. 850 bp or 500 bp) or the number of fragment analyses, the "Kostenstelle" as well as the order number and have it signed by the responsible project manager.
We can only process orders that reach us with a signed "Blockzettel".
c) DNA samples: Please label your sample or primer containers legibly. Please also ensure that the names on the order form and on the sample container match.
After submitting your data using the web-based input mask, you will be given an order number.
You can print out this page for documentation purposes. Please note the order number on the sample bag or sample box.
You will be automatically informed by email about the completion of the order and requested to transfer your results from our server to your own computer via the menu in the navigation bar (only the download of the compressed "zip" file is possible).
Please also note the comments that you will receive with your sequence data. This information can be important for the interpretation of your data and may also contain tips for the success of further sequencing.
We keep your sequences on our server for at least a year so that you can also access the results of older orders. However, we only keep your (DNA) samples for about 1 week!
Important: Your DNA samples may be subject to security level S1 at most.
The exact concentration and the quality of your template (plasmids or PCR products) determine the success of the sequencing reaction. The ratio of the optical density (OD) at 260 and 280 nm (OD260 / OD280) should be between 1.8 and 2.0.
The DNA should be dissolved in the purest water or 10 mM Tris / HCl pH 8. Under no circumstances should the DNA be included in TE [Tris / EDTA] for sequencing.
Since we carry out the analyzes in laboratories with security level S1, your samples must never be subject to a higher security level!
When purifying plasmid DNA for sequencing, it is important to ensure efficient separation of proteins, genomic DNA, RNA, salts and solvents, e.g. with commercial kits, in order to obtain high-quality sequencing results (high reading range, good data quality).
b) PCR products
PCR fragments can be sequenced without prior subcloning, however, it is essential to purify the PCR fragments to separate the PCR components (primers, nucleotides, buffers) prior to sequencing. This can be done with commercially available kits, column-based purification kits for PCR fragments, kits for the elution of fragments from agarose gels or by specific enzymatic digestion of the primers.
|PCR-Produkt||< 100 bp||bis 2,5 ng / µl||ca. 10 ng|
|100 bp - 1 kb||bis 5 ng / µl||50 ng|
|> 1 kb||20 - 50 ng / µl||400 ng|
|Plasmid||< 5 kb||100 - 250 ng / µl||> 500 ng|
|5 kb - 15 kb||150 - 600 ng / µl||> 800 ng|
|> 15 kb||> 600 ng / µl||> 1,5 µg|
|Primer||10 pmol / µl||5 µl|
Please give us sufficient amounts of DNA so that we can carry out optimization or control reactions if necessary.
If your samples reach us before 9 a.m., you will usually receive the results on the following working day if the sequencing is runs fine. If you have particularly important and urgent samples, these samples can also be processed within 4 hours (XPress service).
You can choose between "long read" analyses with up to 1000 bp and "short read" analyses with approx. 500 bp sequence information. Please clearly indicate the desired order category (range of services) on the order form / settlement slip.
Important: "long read" and "short read" analyzes cannot be mixed in one order.
You can of course use your own primers for sequencing. Please note:
If we are to use your individual primers, these must be set to 10 pmol / µl (corresponds to 10 µM). Please also let us know the Tm value of your primer if the annealing temperature is different (standard setting> = 50 ° C). When selecting / constructing your individual primer, please note that there should be 30-50 bases between the 3 'end of the primer (start of sequencing) and the first reliably readable bases. Otherwise you may lose sequence information at the 5 'end of your sequence.
We have a large number of standard primers in stock, which we are happy to use for your DNA sequence analysis free of charge.
|Primer||Sequence 5' -> 3'||suitable for:|
|M13-universal||TGTAAAACGACGGCCAGT||pUC und pBluescript Vektoren|
|further primer upon request|
We keep your (DNA) samples for approx. 1 week! Mark your order / sample bag with an appropriate note if you want to collect your sample material promptly. We keep your sequences on our server for at least a year so that you can also access the results of older orders over the long term.
As a result of the DNA sequencing, you will receive two files with the extensions * .ab1 and * .seq for each sequenced sample. Result files of the DNA fragment analysis have the ending * .fsa. These are PC-files. You can open the files with the following freeware programs:
- Applied Biosystems
- Edit View - Freeware (only for Macintosh up to OS 9.2)
- Chromas - Nominal License Fee
- FinchTV (PC, Macintosh, Linux)
- Sequencher - Free Demo
- Sequence Scanner Software
- Seqdata projects:
- DNAStar - Lasergene
- Vector NTI - Free demo
- Staden MAC OSX - Trev - Free for Academic Sites
- Peak Scanner Software v 1.0
Ideally, this is what your results should look like. (Click to enlarge)
This is what your results will look like when no signal is detected
This is what your results will look like if no signal from the sequencing chemistry could be detected.